Journal of Biological Chemistry

Assembly, purification and pre-steady-state kinetic analysis of an active RNA-dependent RNA polymerase elongation complex.

Publication Date: 2012 Feb 2 PMID: 22303022
Authors: Jin, Z. - Leveque, V. - Ma, H. - Johnson, K. A. - Klumpp, K.
Journal: J Biol Chem

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase-RNA complex suitable for mechanistic and structural studies has remained elusive. Here we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B/primer/template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 +/- 3 muM and 16 +/- 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 muM(-1)s(-1). The kinetics of multiple nucleotide incorporation during processive elongation were also determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

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The vital role of pol zeta and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and the recruitment of pol zeta by REV1 to a replication-stalled site.

Publication Date: 2012 Feb 2 PMID: 22303021
Authors: Hashimoto, K. - Cho, Y. - Yang, I. Y. - Akagi, J. I. - Ohashi, E. - Tateishi, S. - de Wind, N. - Hanaoka, F. - Ohmori, H. - Moriya, M.
Journal: J Biol Chem

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG), using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G-->T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol zeta, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol zeta are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the carboxyl terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol zeta mediated the interaction between REV3 and the REV1 C-terminus. These results support the hypothesis that REV1 recruits pol zeta through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol zeta recruitment pathway. Finally, though mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polzeta in a RAD18-dependent manner.

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Novel bacterial lipoprotein structures conserved in low-GC content Gram-positive bacteria are recognized by Toll-like receptor 2.

Publication Date: 2012 Feb 2 PMID: 22303020
Authors: Kurokawa, K. - Ryu, K. H. - Ichikawa, R. - Matsuda, A. - Kim, M. S. - Lee, H. - Chae, J. H. - Shimizu, T. - Saitoh, T. - Kuwano, K. - Akira, S. - Dohmae, N. - Nakayama, H. - Lee, B. L.
Journal: J Biol Chem

Bacterial lipoproteins/lipopeptides inducing host innate immune responses are sensed by mammalian toll-like receptor 2 (TLR2). These bacterial lipoproteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid. The presence of diacylated lipoproteins has been predicted in low-GC content Gram-positive bacteria and mycoplasmas based on the absence of one modification enzyme in their genomes; however, we recently determined triacylated structures in low-GC Gram-positive Staphylococcus aureus, raising questions about the actual lipoprotein structure in other low-GC content Gram-positive bacteria. Here, through intensive MS analyses, we identified a novel and unique bacterial lipoprotein structure containing an N-acyl-S-monoacyl-glyceryl-cysteine (named the lyso structure) from low-GC Gram-positive Enterococcus faecalis, Bacillus cereus, Streptococcus sanguinis, and Lactobacillus bulgaricus. Two of the purified native lyso-form lipoproteins induced pro-inflammatory cytokine production from mice macrophages in a TLR2-dependent and TLR1-independent manner but with a different dependence on TLR6. Additionally, two other new lipoprotein structures were identified. One is the N-acetyl lipoprotein structure containing N-acetyl-S-diacyl-glyceryl-cysteine, which was found in five Gram-positive bacteria, including Bacillus subtilis. The N-acetyl lipoproteins induced the pro-inflammatory cytokines through the TLR-2/6 heterodimer. The other was identified in a mycoplasma strain and is an unusual diacyl lipoprotein structure containing two amino acids before the lipid-modified cysteine residue. Taken together, our results suggest the existence of novel TLR2-stimulating lyso- and N-acetyl-forms of lipoproteins that are conserved in low-GC content Gram-positive bacteria and provide clear evidence for the presence of yet-to-be identified key enzymes involved in the bacterial lipoprotein biosynthesis.

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Curcumin suppresses T cell activation by blocking Ca2+ mobilization and NFAT activation.

Publication Date: 2012 Feb 2 PMID: 22303019
Authors: Kliem, C. - Merling, A. - Giaisi, M. - Kohler, R. - Krammer, P. H. - Li-Weber, M.
Journal: J Biol Chem

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-kB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T-cell-activation-induced Ca2+ mobilization with IC50 = ~12.5 micro M and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity due to different inhibitory mechanisms. Furthermore, since Ca2+ is also the secondary messenger crucial for the TCR-induced NF-kB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-kB activation.

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Constitutive production of catalytic antibodies to a Staphylococcus aureus virulence factor and effect of infection.

Publication Date: 2012 Feb 2 PMID: 22303018
Authors: Brown, E. L. - Nishiyama, Y. - Dunkle, J. W. - Aggarwal, S. - Planque, S. - Watanabe, K. - Csencsits-Smith, K. - Bowden, M. G. - Kaplan, S. L. - Paul, S.
Journal: J Biol Chem

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. The IgG hydrolyzed peptide bonds on the C terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared to uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared to healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed.

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Structural basis for the antifreeze activity of an ice-binding protein from an Arctic yeast.

Publication Date: 2012 Feb 2 PMID: 22303017
Authors: Lee, J. H. - Park, A. K. - Do, H. - Park, K. S. - Moh, S. H. - Chi, Y. M. - Kim, H. J.
Journal: J Biol Chem

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of approximately 25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57 A and 2.43 A resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed beta-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long alpha-helix that packs along one face of the beta-helix) and a C-terminal hydrophobic loop region (243-PFVPAPEVV-251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common beta-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared to other known AFPs and IBPs.

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Determinants of murein hydrolase targeting to the cross wall of Staphylococcus aureus peptidoglycan.

Publication Date: 2012 Feb 2 PMID: 22303016
Authors: Frankel, M. B. - Schneewind, O.
Journal: J Biol Chem

Cells of eukaryotic or prokaryotic origin express proteins with LysM domains that associate with the cell wall envelope of bacteria. The molecular properties that enable LysM domains to interact with microbial cell walls are not yet established. Staphylococcus aureus, a spherical microbe, secretes two murein hydrolases with LysM domains, Sle1 and LytN. We show here that the LysM domains of Sle1 and LytN direct murein hydrolases to the staphylococcal envelope in the vicinity of the cross wall, the mid-cell compartment for peptidoglycan synthesis. LysM domains associate with the repeating disaccharide beta-N-acetylmuramic acid- (1-->4)-beta-N-acetylglucosamine of staphylococcal peptidoglycan. Modification of N-acetylmuramic acid with wall teichoic acid (WTA), a ribitol-phosphate polymer tethered to murein linkage units, prevents LysM domain binding to peptidoglycan. The localization of LytN and Sle1 to the cross wall is abolished in staphylococcal tagO mutants, which are defective for WTA synthesis. We propose a model whereby the LysM domain ensures septal localization of LytN and Sle1 followed by processive cleavage of peptidoglycan, thereby exposing new LysM binding sites in the cross wall and separating bacterial cells.

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The GABRB3 mutation, G32R, associated with childhood absence epilepsy, alters alpha1beta3gamma2L GABAA receptor expression and channel gating.

Publication Date: 2012 Feb 2 PMID: 22303015
Authors: Gurba, K. N. - Hernandez, C. C. - Hu, N. - Macdonald, R. L.
Journal: J Biol Chem

A GABAA receptor beta3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three beta3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of beta3 subunits. The mutation had three major effects on GABAA receptors. First, coexpression of beta3(G32R) subunits with alpha1 or alpha3 and gamma2L subunits in HEK293T cells reduced surface expression of gamma2L subunits and increased surface expression of beta3 subunits, suggesting a partial shift from ternary alphabeta3gamma2L receptors to binary alphabeta3 and homomeric beta3 receptors. Second, beta3(G32R) subunits were more likely than beta3 subunits to be N-glycosylated at Asn 33,but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, alpha1beta3(G32R)gamma2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because gamma2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at N33). Single channel recording revealed that alpha1beta3(G32R)gamma2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the beta3 subunit N-terminal alpha-helix in receptor assembly and gating.

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The interconnection of salt induced hydrophobic compaction and secondary structure formation depends on solution conditions: revisiting early events of protein folding at single molecule resolution.

Publication Date: 2012 Feb 2 PMID: 22303014
Authors: Haldar, S. - Chattopadhyay, K.
Journal: J Biol Chem

What happens in the early stage of protein folding remains an interesting unsolved problem. Rapid kinetics measurements with cytochrome c using sub-millisecond continuous flow mixing devices suggest simultaneous formation of a compact collapsed state and secondary structure. These data seem to indicate that collapse formation is guided by specific short and long-range interactions (hetero-polymer collapse). A contrasting interpretation has also been proposed which suggests that the collapse formation is rapid, non-specific and a trivial solvent related compaction, which could as well be observed by a homo-polymer (homo-polymer collapse). We address this controversy using fluorescence correlation spectroscopy (FCS), which enables us to monitor the salt induced compaction accompanying collapse formation and the associated time constant directly at single molecule resolution. In addition, we follow the formation of secondary structure using far UV circular dichroism (CD). The data presented here suggest that both these models (homo-polymer and hetero-polymer) could be applicable depending on the solution conditions. For example, the formation of secondary structure and compact state is not simultaneous in aqueous buffer. In aqueous buffer, formation of the compact state occurs through a two-state co-operative transition following hetero-polymer formalism while secondary structure formation takes place gradually. In contrast, in the presence of urea, a compaction of the protein radius occurs gradually over an extended range of salt concentration following homo-polymer formalism. The salt induced compaction and the formation of secondary structure take place simultaneously in the presence of urea.

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Two distinct states of Escherichia coli cells that overexpress the recombinant heterogeneous beta-galactosidase.

Publication Date: 2012 Feb 2 PMID: 22303013
Authors: Zhao, Y. - He, W. - Liu, W. F. - Liu, C. C. - Feng, L. K. - Sun, L. - Yan, Y. B. - Hang, H. Y.
Journal: J Biol Chem

The mechanism by which inclusion bodies form is still not well understood, partly because the dynamic processes of the inclusion body formation and its solubilisation have hardly been investigated at an individual cell level, and so the important detailed information has not been acquired for the mechanism. In this study, we investigated the in vivo folding and aggregation of Aspergillus phoenicis beta-D-galactosidase fused to a red fluorescence protein in individual E. coli cells. The folding status and expression level of the recombinant beta-D-galactosidase at an individual cell level was analyzed by flow cytometry in combination with transmission electron microscopy and Western blotting. We found that individual E. coli cells fell into two distinct states, one containing only inclusion bodies accompanied with low galactosidase activity and the other containing the recombinant soluble galactosidase accompanied with high galactosidase activity. The majority of the E. coli cells in the later state possessed no inclusion bodies. The two states of the cells were shifted to a cell state with high enzyme activity by culturing the cells in IPTG-free medium after an initial protein expression induction in IPTG-containing medium. This shift of the cell population status took place without the level change of the beta-D-galactosidase protein in individual cells, indicating that the factor(s) besides the crowdedness of the recombinant protein play a major role in the cell state ransition. These results shed new light on the mechanism of inclusion body formation and will facilitate the development of new strategies in improving recombinant protein quality.

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Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).

Publication Date: 2012 Feb 1 PMID: 22303012
Authors: Wang, W. - Linsdell, P.
Journal: J Biol Chem

The CFTR chloride channel is a member of the ABC protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between ''outwardly facing'' and ''inwardly facing'' conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large MTSES molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for L102 (first transmembrane region) and T338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the channel's nucleotide binding domains. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional (''vestigial'') gates within the CFTR permeation pathway.

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Crystal structure of a mitochondrial fission complex reveals a scaffolding function for the Mitochondrial Division 1 (Mdv1) coiled coil.

Publication Date: 2012 Feb 1 PMID: 22303011
Authors: Zhang, Y. - Chan, N. C. - Ngo, H. B. - Gristick, H. - Chan, D. C.
Journal: J Biol Chem

The mitochondrial fission machinery is best understood in the yeast Saccharomyces cerevisiae, where Fis1, Mdv1, and Dnm1 are essential components. Fis1 is a mitochondrial outer membrane protein that recruits the dynamin-related GTPase Dnm1 during the fission process. This recruitment occurs via Mdv1, which binds both Fis1 and Dnm1 and therefore functions as a molecular adaptor linking the two molecules. Mdv1 has a modular structure, consisting of an N-terminal extension (NTE) that binds Fis1, a central coiled coil for dimerization, and a C-terminal WD40 repeat region that binds Dnm1. We have solved the crystal structure of a dimeric Mdv1/Fis1 complex that contains both the NTE and coiled coil regions of Mdv1. Consistent with previous studies, Mdv1 binds Fis1 through a U-shaped helix-loop-helix motif, and dimerization of the Mdv1/Fis1 complex is mediated by the anti-parallel coiled coil of Mdv1. However, the complex is surprisingly compact and rigid due to two additional contacts mediated by the surface of the Mdv1 coiled coil. The coiled coil packs against both Fis1 and the second helix of the Mdv1 helix-loop-helix motif. Mutational analyses show that these contacts are important for mitochondrial fission activity. These results indicate that the unusually long Mdv1 coiled coil, in addition to dimerization, serves a scaffolding function to stabilize the Mdv/Fis1 complex.

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During replication stress Non-Smc-Element 5 is required for Smc5/6 complex functionality at stalled forks.

Publication Date: 2012 Feb 2 PMID: 22303010
Authors: Bustard, D. E. - Menolfi, D. - Jeppsson, K. - Ball, L. G. - Dewey, S. C. - Shirahige, K. - Sjogren, C. - Branzei, D. - Cobb, J. A.
Journal: J Biol Chem

The Smc5/6 complex belongs to the Structural Maintenance of Chromosomes (SMC) family that also includes cohesin and condensin. In Saccharomyces cerevisiae the Smc5/6 complex contains six essential Non-Smc-Elements (Nse1-6). Very little is known about how these additional elements contribute to complex function except for Nse2/Mms21, which is an E3 SUMO ligase important for Smc5 sumoylation. Characterization of two temperature sensitive mutants, nse5-ts1 and nse5-ts2, demonstrates the importance of Nse5 within the Smc5/6 complex for its stability and functionality at forks during hydroxyurea (HU)-induced replication stress. Both NSE5 alleles showed a marked reduction in Smc5 sumoylation to levels lower than those observed with mms21-11, a mutant of Mms21 that is deficient in SUMO ligase activity. However, a phenotypic comparison of nse5-ts1 and nse5-ts2 revealed a separation of importance between Smc5 sumoylation and the function of the Smc5/6 complex during replication. Only cells carrying the nse5-ts1 allele exhibited defects such as dissociation of the replisome from stalled forks, the formation of fork-associated homologous recombination (HR) intermediates, and HU sensitivity that is additive with mms21-11. These defects are attributed to a failure in Smc5/6 localization to forks in nse5-ts1 cells. Overall, these data support the premise that Nse5 is important for vital interactions between components within the Smc5/6 complex, which is a prerequisite for its functionality during replication stress.

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Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to the RAB11-dependent slow recycling pathway.

Publication Date: 2012 Feb 2 PMID: 22303009
Authors: Madsen, K. L. - Thorsen, T. S. - Rahbek-Clemmensen, T. - Eriksen, J. - Gether, U.
Journal: J Biol Chem

The scaffolding protein PICK1 contains an N-terminal PDZ (PSD-95/Discs-large/ZO-1) domain and a central lipid binding BAR (Bin/amphiphysin/Rvs) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK-293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1 and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1 binding DAT C-terminus to the beta2-adrenergic receptor (beta2AR), which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11 positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain dependent manner. In contrast, transfer of the DAT C-terminus to the delta-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters and any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11 mediated recycling pathway.

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Bacterial degradation of benzoate: Cross-regulation between aerobic and anaerobic pathways.

Publication Date: 2012 Feb 2 PMID: 22303008
Authors: Valderrama, J. A. - Durante-Rodriguez, G. - Blazquez, B. - Garcia, J. L. - Carmona, M. - Diaz, E.
Journal: J Biol Chem

We have studied for the first time the transcriptional regulatory circuit that controls the expression of the box genes encoding the aerobic hybrid pathway used to assimilate benzoate via coenzyme A (CoA) derivatives in bacteria. The promoters responsible for the expression of the box cluster in the beta-proteobacterium Azoarcus sp., their cognate transcriptional repressor, the BoxR protein, and the inducer molecule (benzoyl-CoA) have been characterized. The BoxR protein shows a significant sequence identity to the BzdR transcriptional repressor that controls the bzd genes involved in the anaerobic degradation of benzoate. Since the boxR gene is present in all box clusters so far identified in bacteria, the BoxR/benzoyl-CoA regulatory system appears to be a widespread strategy to control this aerobic hybrid pathway. Interestingly, the paralogous BoxR and BzdR regulators act synergistically to control the expression of the box and bzd genes. This cross-regulation between anaerobic and aerobic pathways for the catabolism of aromatic compounds has never been shown before, and it may reflect a biological strategy to increase the cell fitness in organisms that survive in environments subject to changing oxygen concentrations.

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Direct Role for proliferating cell nuclear antigen (PCNA) in substrate recognition by the E3 Ubiquitin ligase CRL4-Cdt2.

Publication Date: 2012 Feb 2 PMID: 22303007
Authors: Havens, C. G. - Shobnam, N. - Guarino, E. - Centore, R. C. - Zou, L. - Kearsey, S. E. - Walter, J. C.
Journal: J Biol Chem

The E3 ubiquitin ligase Cullin-Ring Ligase 4- Cdt2 (CRL4-Cdt2) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21 and Set8. CRL4-Cdt2 substrates contain a 'PIP degron,' which consists of a canonical Proliferating Cell Nuclear Antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4-Cdt2 for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4-Cdt2 substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4-Cdt2 recruitment to chromatin. Our data show that the interaction of CRL4-Cdt2 with substrates requires molecular determinants not only in the substrate degron, but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.

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Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by Heme Responsive Gene-2.

Publication Date: 2012 Feb 2 PMID: 22303006
Authors: Chen, C. - Samuel, T. K. - Krause, M. - Dailey, H. A. - Hamza, I.
Journal: J Biol Chem

The roundworm Caenorhabditis elegans is a heme auxotroph that requires the coordinated actions of HRG-1 heme permeases to transport environmental heme into the intestine and HRG-3, a secreted protein, to deliver intestinal heme to other tissues including the embryo. Here we show that heme homeostasis in the extraintestinal hypodermal tissue is facilitated by the transmembrane protein HRG-2. Systemic heme deficiency upregulates hrg-2 mRNA expression over 200-fold in the main body hypodermal syncytium hyp 7. HRG-2 is a type I membrane protein which binds heme and localizes to the endoplasmic reticulum and apical plasma membrane. Cytochrome heme profiles are aberrant in HRG-2 deficient worms, a phenotype that is partially suppressed by heme supplementation. Heme-deficient yeast strain, ectopically expressing worm HRG-2, reveal significantly improved growth at submicromolar concentrations of exogenous heme. Taken together, our results implicate HRG-2 as a facilitator of heme utilization in the C. elegans hypodermis and provide a mechanism for regulation of heme homeostasis in an extraintestinal tissue.

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Structural requirements of membrane phospholipids for M-type potassium channel activation and binding.

Publication Date: 2012 Feb 1 PMID: 22303005
Authors: Telezhkin, V. - Reilly, J. M. - Thomas, A. M. - Tinker, A. - Brown, D. A.
Journal: J Biol Chem

M-channels are voltage-gated potassium channels that regulate cell excitability. They are heterotrameric assemblies of Kv7.2 and Kv7.3 subunits. Their opening requires the presence of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate,PI(4,5)P(2) . However, the specificity of PI(4,5)P(2) as a binding and activating ligand is unknown. Here we tested the ability of different phosphoinositides and lipid phosphates to activate or bind to M-channel proteins. Activation of functional channels was measured in membrane patches isolated from cells co-expressing Kv7.2 and Kv7.3 subunits. Channels were activated to similar extents (maximum open probability ~0.8 at 0 mV) by 0.1 to 300 muM of dioctanoyl (diC(8))homologs of the three endogenous phosphoinositides PI(4)P, PI(4,5)P(2) and PI(3,4,5)P(3), sensitivity increasing with increasing number of phosphates. Non-acylated inositol phosphates had no effect up to 100 micromolar. Channels were also activated with increasing efficacy by 1-300 muM of the monoacyl monophosphates fingolimod phosphate, sphingosine-1-phosphate and lysophosphatidic acid, but not by phosphate-free fingolimod or sphingosine, nor by phosphate-masked phosphatidylcholine or phosphatidylglycerol. An overlay assay confirmed that a fusion protein containing the full-length C-terminus of Kv7.2 could bind to a broad range of phosphoinositides and phospholipids. A mutated Kv7.2 C-terminal construct with reduced sensitivity to PI(4,5)P showed significantly less binding to most polyphosphoinositides. It is concluded that M-channels bind to, and are activated by, a wide range of lipid phosphates, with a minimal requirement for an acyl chain and a phosphate head-group. In this, they more closely resemble Kir6.2 inward-rectifier potassium channels than the more PI(4,5)P(2-)specific Kir2 channels. Notwithstanding, the data also support the view that the main endogenous activator of M-channels is PI(4,5)P2.

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Phosphorylation of Sar1b releases the liver fatty acid binding protein from a multi-protein complex in intestinal cytosol enabling it to bind to the ER and bud the Pre-chylomicron transport vesicle.

Publication Date: 2012 Feb 1 PMID: 22303004
Authors: Siddiqi, S. - Mansbach, C. M. 2nd
Journal: J Biol Chem

Native cytosol requires ATP to initiate the budding of the pre-chylomicron transport vesicle from intestinal ER. When FABP1 alone is used, no ATP is needed. Here we test the hypothesis that in native cytosol, FABP1 is present in a multi-protein complex that prevents FABP1 binding to the ER unless the complex is phosphorylated. We found on chromatography of native intestinal cytosol over a Sephacryl S-100 HR column, that FABP1 (14 kDa) eluted in a volume suggesting a 75 kDa protein complex. Native gel PAGE showed similar results. An anti-FABP1 antibody pull down revealed 3 other proteins bound to FABP1. The FABP1 containing fractions from the Sephacryl column were chromatographed over an anti-FABP1 antibody adsorption column. FABP1, Sar1b, Sec13 and SVIP were co-eluted from the column. The 4 proteins of the complex had a total Mr of 77 kDa and migrated on native PAGE at 75 kDa. When the complex was incubated with intestinal ER, there was no increase in FABP1 ER binding. However, when the complex member Sar1b was phosphorylated by PKCzeta the complex completely disassembled into its component parts that now migrated at their monomer Mr on native PAGE. FABP1, now freed from the complex, was able to bind to intestinal ER and generate PCTV. No increase in ER binding or PCTV generation was observed in the absence of PKCzeta or ATP. We conclude that phosphorylation of Sar1b disrupts the FABP1 containing 75 kDa protein complex in cytosol enabling it to bind to the ER and generate PCTV.

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Platelet-activating factor receptor agonists mediate Xeroderma Pigmentosum A photosensitivity.

Publication Date: 2012 Feb 1 PMID: 22303003
Authors: Yao, Y. - Harrison, K. A. - Al-Hassani, M. - Murphy, R. C. - Rezania, S. - Konger, R. L. - Travers, J. B.
Journal: J Biol Chem

To date, oxidized glycerol-phosphocholines (Ox-GPC) with Platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPC play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein Xeroderma Pigmentosum complementation group A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor, ultraviolet B radiation (UVB), resulted in increased reactive oxygen species and PAF-receptor (PAF-R) agonistic activity in comparison to gene-corrected cells. The UVB-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison to control mice. The increased UVB-mediated skin inflammation and TNF-alpha production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chained Ox-GPC along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPC in the pathophysiology of XPA photosensitivity.

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Sphingolipid-modulated exosome secretion promotes the clearance of amyloid-beta by microglia.

Publication Date: 2012 Feb 2 PMID: 22303002
Authors: Yuyama, K. - Sun, H. - Mitsutake, S. - Igarashi, Y.
Journal: J Biol Chem

Amyloid beta-peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular Abeta is associated with exosomes, small membrane vesicles of endosomal origin, although the fate of Abeta in association with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular Abeta, i. e., exosomes drive conformational changes in Abeta to form nontoxic amyloid fibrils, and promote uptake of Abeta by microglia. The Abeta internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed Abeta incorporation into microglia. In addition, we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell experiments, upregulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced Abeta uptake into microglial cells and significantly decreased extracellular levels of Abeta. Our findings indicate a novel mechanism responsible for clearance of Abeta through its association with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD.

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A two-step mechanism for modifier of transcription 1 (Mot1)-catalyzed displacement of TATA-binding protein (TBP) from DNA.

Publication Date: 2012 Feb 1 PMID: 22298788
Authors: Moyle-Heyrman, G. - Viswanathan, R. - Widom, J. - Auble, D. T.
Journal: J Biol Chem

The TATA box binding protein (TBP) is a central component of the transcription preinitiation complex (PIC) and its occupancy at a promoter is correlated with transcription levels. The TBP-promoter DNA complex contains sharply bent DNA and its interaction lifetime is limited by the ATP-dependent TBP displacement activity of the Snf2/Swi2 ATPase Mot1. Several mechanisms for Mot1 action have been proposed, but how it catalyzes TBP removal from DNA is unknown. To better understand the Mot1 mechanism, native gel electrophoresis and fluorescence resonance energy transfer (FRET) were used to determine how Mot1 affects the trajectory of DNA in the TBP-DNA complex. Strikingly, in the absence of ATP, Mot1 acts to unbend DNA, even while TBP remains closely associated with the DNA in a stable Mot1-TBP-DNA ternary complex. Interestingly, and in contrast to full-length Mot1, the isolated Mot1 ATPase domain binds DNA, and its affinity for DNA is nucleotide-dependent, suggesting parallels between the Mot1 mechanism and DNA translocation-based mechanisms of chromatin remodeling enzymes. Based on these findings, a model is presented for Mot1 that links a DNA conformational change with ATP-induced DNA translocation.

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SAPSR1 is a bidentate anchor for targeting protein phosphatase-6 to DNA-PK.

Publication Date: 2012 Feb 1 PMID: 22298787
Authors: Hosing, A. S. - Valerie, N. C. - Dziegielewski, J. - Brautigan, D. L. - Larner, J. M.
Journal: J Biol Chem

DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining (NHEJ) pathway. DNA-PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation (IR)-induced DNA damage, and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1-326 and 522-700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1-326, not 522-700, eliminated interaction of R1 with DNA-PK. We assign 1-326 as the dominant domain and 522-700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruit PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to IR and other genotoxic agents.

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The role of metalloproteases in vaccinia virus epitope processing for transporter associated with antigen processing (TAP)-independent human leukocyte antigen (HLA)-B7 class I antigen presentation.

Publication Date: 2012 Feb 1 PMID: 22298786
Authors: Lorente, E. - Garcia, R. - Mir, C. - Barriga, A. - Lemonnier, F. A. - Ramos, M. - Lopez, D.
Journal: J Biol Chem

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with non-functional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections.

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Under-sulfation of heparan sulfate restricts the differentiation potential of mouse embryonic stem cells.

Publication Date: 2012 Feb 1 PMID: 22298785
Authors: Forsberg, M. - Holmborn, K. - Kundu, S. - Dagalv, A. - Kjellen, L. - Forsberg-Nilsson, K.
Journal: J Biol Chem

Heparan sulfate proteglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The NDST (glucosaminyl N-deacetylase/N-sulfotransferase) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts, but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also GATA4, a primitive endoderm marker, was expressed by these cells. Addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step towards differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.

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Piceatannol, a natural polyphenolic stilbene, inhibits adipogenesis via modulation of mitotic clonal expansion and insulin receptor-dependent insulin signaling in the early phase of differentiation.

Publication Date: 2012 Jan 31 PMID: 22298784
Authors: Kwon, J. Y. - Seo, S. G. - Heo, Y. S. - Yue, S. - Cheng, J. X. - Lee, K. W. - Kim, K. H.
Journal: J Biol Chem

Piceatannol, a natural stilbene, is an analogue and a metabolite of resveratrol. Despite a well-documented health benefit of resveratrol in intervention of the development of obesity, the role of piceatannol in the development of adipose tissue and related diseases is unknown. Here we sought to determine the function of piceatannol in adipogenesis and elucidate the underlying mechanism. We show that piceatannol inhibits adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner at non-cytotoxic concentrations. This anti-adipogenic property of piceatannol was largely limited to the early event of adipogenesis. In the early phase of adipogenesis, piceatannol-treated preadipocytes displayed a delayed cell cycle entry into G2/M phase at 24 hr after initiation of adipogenesis. Furthermore, the piceatannol-suppressed mitotic clonal expansion was accompanied by reduced activation of insulin-signaling pathway. Piceatannol dose-dependently inhibited differentiation cocktail-induced phosphorylation of insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/Akt pathway in the early phase of adipogenesis. Moreover, we showed that piceatannol is an inhibitor of IR kinase and phosphatidylinositol 3-kinase (PI3K). Our kinetics study of IR kinase further identified a K(m) value for ATP of 57.8 muM and a K(i) value for piceatannol of 28.9 muM. We also showed that piceatannol directly binds to IR and inhibits IR kinase activity in a mixed non-competitive manner to ATP, through which piceatannol appears to inhibit adipogenesis. Taken together, our study reveals an anti-adipogenic function of piceatannol and highlights IR and its downstream insulin signaling as novel targets for piceatannol in the early phase of adipogenesis.

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Function of TRPV4 as a mechanical transducer in flow-sensitive segments of the renal collecting duct system.

Publication Date: 2012 Feb 1 PMID: 22298783
Authors: Berrout, J. - Jin, M. - Mamenko, M. - Zaika, O. - Pochynyuk, O. - O'Neil, R. G.
Journal: J Biol Chem

The TRPV4 Ca2+-permeable channel is sensitive to mechanical stimuli. In the current study we have employed immunocytochemical staining in kidney slices and functional assessments (Ca2+ imaging) in isolated, split-open, tubule segments to define TRPV4 sites of expression and flow-dependent function in the collecting duct system. Staining patterns revealed strong expression of TRPV4 along the entire collecting duct system with highest levels at the apical (luminal)/sub-apical region of the principal cells (PC), the dominant cell type, with more diffuse staining in intercalated cells (IC). Using fluorescence Ca2+ imaging and the selective TRPV4 agonist, GSK1016790A, we demonstrated functional TRPV4 channels in PC and IC of split-opened cortical collecting ducts (CCD) and connecting tubules (CNT). The agonist was ineffective in inducing a rise in [Ca2+]i in the absence of extracellular Ca2+ or in tubules from TRPV4 deficient animals. Most importantly, a 10-fold elevation in luminal (apical) fluid flow induced a rapid and sustained influx of Ca2+ that was abolished by the TRPV channel inhibitor, ruthenium red, or in tubules isolated from TRPV4 deficient animals. We concluded that TRPV4 is highly expressed along the entire collecting duct system where it appears to function as a sensor/transducer of flow-induce mechanical stresses.

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P2Y2 receptor-mediated lymphotoxin-alpha (LTA) secretion regulates intercellular cell adhesion molecule (ICAM)-1 expression in vascular smooth muscle cells.

Publication Date: 2012 Feb 1 PMID: 22298782
Authors: Seye, C. I. - Agca, Y. - Agca, C. - Derbigny, W.
Journal: J Biol Chem

The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y2R nucleotide receptor (P2Y2R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y2R KO SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi, suggesting that P2Y2R R/filamin mediated LTA secretion is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y2R deficient in LTA secretion.These data suggest that P2Y2R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.

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alpha-Catenin inhibits beta-catenin-Tcf/Lef transcriptional activity and collagen type II expression in articular chondrocytes through formation of a Gli3R/alpha-catenin/beta-catenin ternary complex.

Publication Date: 2012 Feb 1 PMID: 22298781
Authors: Rhee, J. - Ryu, J. H. - Kim, J. H. - Chun, C. H. - Chun, J. S.
Journal: J Biol Chem

Chondrocytes, a unique cell type in cartilage tissue, are responsible for the regulation of anabolic and catabolic homeostasis in cartilage-specific extracellular matrix synthesis. Activation of Wnt/beta-catenin signaling induces dedifferentiation of articular chondrocytes, resulting in suppression of type II collagen expression. We have previously shown that alpha-catenin inhibits beta-catenin-Tcf/Lef (T-cell factor/lymphoid-enhancing factor) transcriptional activity in articular chondrocytes with a concomitant recovery of type II collagen expression. In the current study, we elucidated the mechanism underlying this inhibition of beta-catenin-Tcf/Lef transcriptional activity by alpha-catenin, showing that it requires direct interaction between alpha-catenin and beta-catenin. We further showed that it involves recruitment of Gli3R, the short transcription-repressing form of the transcription factor Gli3, to beta-catenin by alpha-catenin. The resulting inhibition of beta-catenin transcriptional activity leads to increased expression of type II collagen. Gli3R and alpha-catenin actions are co-dependent: both are necessary for the observed inhibitory effects on beta-catenin transcriptional activity. Reducing Gli3R expression levels through activation of Indian Hedgehog (Ihh) signaling is also sufficient to activate beta-catenin transcriptional activity, suggesting that the ternary complex, Gli3R/alpha-catenin/beta-catenin, mediates Ihh-dependent activation of Wnt/beta-catenin signaling in articular chondrocytes. Collectively, this study shows that alpha-catenin functions as a nuclear factor that recruits the transcriptional repressor Gli3R to alpha-catenin to inhibit beta-catenin transcriptional activity and prevent dedifferentiation of articular chondrocytes.Finally, osteoarthritic cartilage showed elevated levels of beta-catenin and decreased levels of alpha-catenin and Gli3R, suggesting that decreased levels of alpha-catenin and Gli3R levels contribute to increased beta-catenin transcriptional activity during osteoarthritic cartilage destruction.

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The extreme C-terminus of the bacterial cytoskeletal protein FtsZ plays a fundamental role in assembly independent of modulatory proteins.

Publication Date: 2012 Feb 1 PMID: 22298780
Authors: Buske, P. J. - Levin, P. A.
Journal: J Biol Chem

Bacterial cell division typically requires assembly of the cytoskeletal protein FtsZ into a ring (Z-ring) at the nascent division site that serves as a foundation for assembly of the division apparatus. High-resolution imaging suggests the Z-ring consists of short, single stranded polymers held together by lateral interactions. Several proteins implicated in stabilizing the Z-ring enhance lateral interactions between FtsZ polymers in vitro. Here we report that residues at the C-terminus of B. subtilis FtsZ (CTV for C-terminal variable region) are both necessary and sufficient for stimulating lateral interactions in vitro in the absence of modulatory proteins. Swapping the 6-residue CTV from B. subtilis FtsZ with the 4-residue CTV from E. coli FtsZ completely abolished lateral interactions between chimeric B. subtilis FtsZ polymers. The E. coli FtsZ chimera readily formed higher order structures normally seen in only the presence of molecular crowding agents. CTV mediated lateral interactions are important for integrity of the Z-ring, as B. subtilis cells expressing the B. subtilis FtsZ chimera had a low frequency of FtsZ ring formation and a high degree of filamentation relative to wild type cells. Site-directed mutagenesis of the B. subtilis CTV suggests electrostatic forces are an important determinant of lateral interaction potential.

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Bivalent carbohydrate binding is required for biological activity of CNL, the LacdiNAc (GalNAcbeta1-4GlcNAc)-specific lectin from basidiomycete Clitocybe nebularis.

Publication Date: 2012 Feb 1 PMID: 22298779
Authors: Pohleven, J. - Renko, M. - Magister, S. - Smith, D. F. - Kuenzler, M. - Strukelj, B. - Turk, D. - Kos, J. - Sabotic, J.
Journal: J Biol Chem

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis, in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcbeta1-4GlcNAc, LacdiNAc, LDN) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LDN, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers comprises a single ricin B lectin domain with its beta-trefoil fold and one carbohydrate-binding site. In order to study the mode of CNL action, a non-sugar-binding mutant and non-dimerizing monovalent mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, while the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.

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cAMP induces stromal interaction molecule 1 (STIM1) Puncta but neither orai1 clustering nor store-operated Ca2+ entry (SOCE) in islet cells.

Publication Date: 2012 Feb 1 PMID: 22298778
Authors: Tian, G. - Tepikin, A. V. - Tengholm, A. - Gylfe, E.
Journal: J Biol Chem

The events leading to the activation of store-operated Ca(2+) entry (SOCE) involve Ca(2+) depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca(2+) sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane (PM) where it binds to the Ca(2+) channel protein Orai1 to activate Ca(2+) influx. Using confocal and total internal reflection fluorescence microscopy we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing beta and glucagon-secreting alpha cells within intact mouse and human pancreatic islets. ER Ca(2+) depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the PM and activated SOCE. Glucose, which promotes Ca(2+) store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in alpha than in beta cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in alpha cells and retranslocation in beta cells involving rise and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and Exchange protein directly activated by cAMP (Epac). However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1 and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.

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Characterization of the heparin binding site of tissue transglutaminase: its importance in the enzyme's cell surface targeting, matrix deposition and cell signalling.

Publication Date: 2012 Feb 1 PMID: 22298777
Authors: Wang, Z. - Collighan, R. J. - Pytel, K. - Rathbone, D. L. - Li, X. - Griffin, M.
Journal: J Biol Chem

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCalpha, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

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Insights into the mechanism of glucokinase activation: observation of multiple distinct protein conformations.

Publication Date: 2012 Feb 1 PMID: 22298776
Authors: Liu, S. - Ammirati, M. J. - Song, X. - Knafels, J. D. - Zhang, J. - Greasley, S. E. - Pfefferkorn, J. A. - Qiu, X.
Journal: J Biol Chem

Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and ones that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKA) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion (GSIS), but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK/glucose complex in a wide-open conformation. To assess conformations of GK in solution, we also carried out small angle X-ray scattering (SAXS) experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared to wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKA with reduced hypoglycemic risk.

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FoxO1 cooperates with ATF4 in osteoblasts to control glucose homeostasis.

Publication Date: 2012 Feb 1 PMID: 22298775
Authors: Kode, A. - Mosialou, I. - Silva, B. C. - Joshi, S. - Ferron, M. - Rached, M. T. - Kousteni, S.
Journal: J Biol Chem

The Forkhead transcription factor FoxO1 inhibits through its expression in osteoblasts beta-cell proliferation, insulin secretion and sensitivity. At least part of the FoxO1 metabolic functions result from its ability to suppress the activity of osteocalcin, an osteoblast-derived hormone favoring glucose metabolism and energy expenditure. In searching for mechanisms mediating the metabolic actions of FoxO1 we focused on ATF4, because this transcription factor also affects glucose metabolism through its expression in osteoblasts. We show here that FoxO1 co-localizes with ATF4 in the osteoblast nucleus, and physically interacts with and promotes the transcriptional activity of ATF4. Genetic experiments demonstrate that FoxO1 and ATF4 cooperate to increase glucose levels and decrease glucose tolerance. These effects result from a synergistic effect of the two transcription factors to suppress the activity of osteocalcin through upregulating expression of the phosphatase catalyzing osteocalcin inactivation. As a result, insulin production by beta-cells and insulin signaling in the muscle, liver and white adipose tissue is compromised and fat weight increases by the FoxO1/ATF4 interaction. Taken together these observations demonstrate that FoxO1 and ATF4 cooperate in osteoblasts to regulate glucose homeostasis.

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The SEC23-SEC31 interface plays a critical role for export of procollagen from the endoplasmic reticulum.

Publication Date: 2012 Feb 1 PMID: 22298774
Authors: Kim, S. D. - Pahuja, K. B. - Ravazzola, M. - Yoon, J. - Boyadjiev, S. A. - Hammamoto, S. - Schekman, R. - Orci, L. - Kim, J.
Journal: J Biol Chem

COPII proteins are essential for exporting most cargo molecules from the endoplasmic reticulum (ER). The membrane-facing surface of the COPII proteins (especially SEC23/24) interacts directly or indirectly with the cargo molecules destined for exit. As we characterized the SEC23A mutations at the SEC31 binding site identified from patients with cranio-lenticulo-sutural dysplasia (CLSD), we discovered that the SEC23-SEC31 interface can also influence cargo selection. Remarkably, M702V SEC23A does not compromise COPII assembly, vesicle size, and packaging of cargo molecules into COPII vesicles that we have tested, but induces accumulation of procollagen in the ER when expressed in normal fibroblasts. We observed that M702V SEC23A activates SAR1B GTPase more than wild-type SEC23A when SEC13/31 is present, indicating that M702V SEC23A causes premature dissociation of COPII from the membrane. Our results indicate that a longer stay of COPII proteins on the membrane is required to cargo procollagen than other molecules and suggest that the SEC23-SEC31 interface plays a critical role in capturing various cargo molecules.

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Heparan sulfate chains of syndecan-1 regulate ectodomain shedding.

Publication Date: 2012 Feb 1 PMID: 22298773
Authors: Ramani, V. C. - Pruett, P. S. - Thompson, C. A. - Delucas, L. D. - Sanderson, R. D.
Journal: J Biol Chem

Matrix metalloproteinases (MMPs) release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by MMPs. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

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Age-related changes in glycosaminoglycans from rat myocardium involve altered capacities to potentiate growth factors functions and heparan sulfate altered sulfation.

Publication Date: 2012 Feb 1 PMID: 22298772
Authors: Huynh, M. B. - Morin, C. - Carpentier, G. - Garcia-Filipe, S. - Talhas-Perret, S. - Barbier-Chassefiere, V. - van Kuppevelt, T. H. - Martelly, I. - Albanese, P. - Papy-Garcia, D.
Journal: J Biol Chem

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from where cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Since most heparin binding growth factors (HBGF) activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated if the aging process can induce changes in the myocardial GAGs composition in rats, and if these changes can affect the activities of particular HBGF known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in left ventricle. Biochemical and immunohistological assessments pointed out heparan sulfates (HS) as the GAGs species increasing with age. ELISA based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB-EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAGs capacities to potentiate FGF-2 while the potentiating effect on VEGF165 was increased, as confirmed by augmented angiogenic cells proliferation in matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with only modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. These functional alterations can be associated to an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

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Organ-Specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases Sulf1 and Sulf2 in Mice.

Publication Date: 2012 Feb 1 PMID: 22298771
Authors: Nagamine, S. - Tamba, M. - Ishimine, H. - Araki, K. - Shiomi, K. - Okada, T. - Ohto, T. - Kunita, S. - Takahashi, S. - Wismans, R. G. - van Kuppevelt, T. H. - Masu, M. - Keino-Masu, K.
Journal: J Biol Chem

Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knockout mice. Significant increases in DeltaUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in DeltaUA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in 8 organs of wild-type mice were highly correlated with increases in DeltaUA2S-GlcNS6S in the corresponding organs of knockout mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knockout mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.

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The kinetics of alpha-globin binding to alpha hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of a hemichrome folding intermediate.

Publication Date: 2012 Feb 1 PMID: 22298770
Authors: Mollan, T. L. - Khandros, E. - Weiss, M. J. - Olson, J. S.
Journal: J Biol Chem

Human Alpha-Hemoglobin Stabilizing Protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A (HbA) by stabilizing free alpha globin. AHSP rapidly binds to ferrous alpha, with association (k'AHSP) and dissociation (kAHSP) rate constants approximately 10 muM-1s-1 and 0.2 s-1, respectively, at pH 7.4, 22 OC. A small slow phase is observed when AHSP binds to excess ferrous alphaCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp29-Pro30 peptide bond in wild-type AHSP because it is absent when alphaCO is mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase is also absent when met(Fe3+)-alpha reacts with wild-type AHSP, suggesting that met-alpha is capable of rapidly binding to either Pro-30 conformer. Both wild-type and Pro-30 substituted AHSPs drive the formation of a met-alpha hemichrome conformation following binding to either met- or oxy(Fe2+)-alpha. The dissociation rate of the met-alpha:AHSP complex (kAHSP approximately 0.002 s-1) is approximately 100-fold slower than that for ferrous-alpha:AHSP complexes, resulting in a much higher affinity of AHSP for met-alpha. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-alpha hemichrome folding intermediates. The low rate of met-alpha dissociation also allows AHSP to have a quality control function by kinetically trapping ferric alpha and preventing its incorporation into less stable mixed valence HbA tetramers. Reduction of AHSP-bound met-alpha allows more rapid release to beta subunits to form stable fully reduced hemoglobin dimers and tetramers.

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Role of epithelial-stem cell interactions during dental cell differentiation.

Publication Date: 2012 Feb 1 PMID: 22298769
Authors: Arakaki, M. - Ishikawa, M. - Nakamura, T. - Iwamoto, T. - Yamada, A. - Fukumoto, E. - Saito, M. - Otsu, K. - Harada, H. - Yamada, Y. - Fukumoto, S.
Journal: J Biol Chem

Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.

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Inducible malondialdehyde pools in zones of cell proliferation and developing tissues in Arabidopsis.

Publication Date: 2012 Feb 1 PMID: 22298768
Authors: Schmid-Siegert, E. - Loscos, J. - Farmer, E. E.
Journal: J Biol Chem

Malondialdehyde (MDA) is a natural and widespread genotoxin. Given its potentially deleterious effects it is of interest to establish the identities of the cells types containing this aldehyde. We used in situ chemical trapping with 2-thiobarbituric acid and mass spectrometry with a deuterated standard to characterize MDA pools in the vegetative phase in Arabidopsis thaliana. In leaves, MDA occurred predominantly in the intracellular compartment of mesophyll cells and was enriched in chloroplasts where it was derived primarily from triunsaturated fatty acids (TFAs). High levels of MDA (most of which was unbound) were found within dividing cells in the root tip cell proliferation zone. The bulk of this MDA did not originate from TFAs. We confirmed the localization of MDA in transversal root sections. In addition to MDA in proliferating cells near the root tip we found evidence for the presence of MDA in pericyle cells. Remodelling of non-TFA-derived MDA pools occurred when seedlings were infected with the fungus Botrytis cinerea. Treatment of uninfected seedlings with mediators of plant stress responses (jasmonic acid or salicylic acid) increased seedling MDA levels over 20-fold. In summary, major pools of MDA are associated with cell division foci containing stem cells. The aldehyde is pathogen-inducible in these regions and its levels are increased by cellular mediators that impact defense and growth.

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Hepatic stellate cell-derived delta-like homolog 1 (DLK1) in liver regeneration.

Publication Date: 2012 Feb 1 PMID: 22298767
Authors: Zhu, N. L. - Asahina, K. - Wang, J. - Ueno, A. - Lazaro, R. - Miyaoka, Y. - Miyajima, A. - Tsukamoto, H.
Journal: J Biol Chem

Hepatic stellate cells (HSCs) undergo myofibroblastic activation in liver fibrosis and regeneration. This phenotypic switch is mechanistically similar to de-differentiation of adipocytes as such the Necdin-Wnt pathway causes epigenetic repression of the master adipogenic gene Ppargamma, to activate HSCs. Now we report delta-like 1 homolog (DLK1) is expressed selectively in HSCs in the adult rodent liver and induced in liver fibrosis and regeneration. Dlk1 knockdown in activated HSCs, causes suppression of Necdin and Wnt, epigenetic de-repression of Ppargamma, and morphologic and functional reversal to quiescent cells. Hepatic Dlk1 expression is induced 40-fold 1 day after partial hepatectomy (PH) in mice. HSCs and hepatocytes (HC) isolated from the regenerating liver show Dlk1 induction in both cell types. In HC and HSC co-culture, increased proliferation and Dlk1 expression by HC from PH are abrogated with anti-DLK1 antibody (Ab). Dlk1 and Wnt10b expression by Sham HC are increased by co-culture with PH HSCs, and these effects are abolished with anti-DLK Ab. A tail-vein injection of anti-DLK1 Ab at 6 hr after PH reduces early HC proliferation and liver growth, accompanied by decreased Wnt10b, non-phosphorylated beta-catenin, p-beta-catenin (S552), cyclins (CyclinD and CyclinA), cyclin dependent kinases (CDK4, and CDK1/2), p-ERK1/2, and p-AKT. In the mouse developing liver, HSC precursors and HSCs express high levels of Dlk1, concomitant with Dlk1 expression by hepatoblasts. These results suggest novel roles of HSC-derived DLK1 in activating HSCs via epigenetic Ppargamma repression and participating in liver regeneration and development in a manner involving the mesenchymal-epithelial interaction.

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Internalization of the proprotein convertase PC7 from the plasma membrane is mediated by a novel motif.

Publication Date: 2012 Jan 30 PMID: 22294700
Authors: Declercq, J. - Meulemans, S. - Plets, E. - Creemers, J. W.
Journal: J Biol Chem

PC7 is a member of the subtilisin-like proprotein convertase family, which is involved in the endoproteolysis of a variety of precursor proteins. Under steady state conditions, PC7 is mainly localized in the trans-Golgi network (TGN), but a small fraction is found at the cell surface. So far no sorting signals for membrane trafficking have been identified in PC7. In this study we have examined the internalization of PC7 from the plasma membrane. Our results show that internalization of PC7 is mediated by clathrin-coated vesicles (CCVs). After inhibition of clathrin-mediated endocytosis using hypertonic conditions or the small molecule inhibitor Pitstop 2, PC7 accumulated at the plasma membrane. Furthermore, PC7 was present in isolated CCVs. To determine the internalization motif, constructs were generated in which parts of the N- and C-terminus of the cytoplasmic tail of PC7 were deleted and chimeric proteins were constructed consisting of the luminal and transmembrane domains of Tac (CD25) and parts of the cytoplasmic domain of PC7. Antibody uptake experiments as well as surface biotinylation experiments demonstrated that the region between A713 and C726 in the cytoplasmic domain of PC7 is essential and sufficient for the internalization of PC7 but not for TGN localization. Individual amino acids in this region were substituted with alanine which identified Pro Leu and Cys as the essential amino acids. In conclusion, internalization of PC7 depends on a short transferable sequence in the cytoplasmic tail which contains the three crucial amino acids "PLC".

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Identification and Characteriation of Nardilysin as a Novel Dimethyl H3K4 Binding Protein Involved in Transcriptional Regulation.

Publication Date: 2012 Jan 30 PMID: 22294699
Authors: Li, J. - Chu, M. - Wang, S. - Chan, D. - Qi, S. - Wu, M. - Zhou, Z. - Li, J. - Nishi, E. - Qin, J. - Wong, J.
Journal: J Biol Chem

Histone methylation on lysine residues is believed to function primarily as docking sites to recruit specific proteins termed as histone code readers or effectors. Each lysine residue can be mono-, di and tri-methylated and different methylation states can have different effect on chromatin function. While an increasing number of proteins has been identified and characterized as specific effectors for methylated histones, very few of the proteins are known to recognize a particular state of methylation. In this study, we identified nardilysin (NRDc), a member of M16 family metalloendopeptidases, as a novel dimethyl-H3K4 (H3K4me2) binding protein. Among three methylated states, NRDc binds preferentially H3K4me2 both in vitro and in vivo. Biochemical purification demonstrated that NRDc interacts with the NCoR/SMRT corepressor complex. We identified target genes repressed by NRDc through microarray. We showed that NRDc is physically associated with and recruits the NCoR complex to the repressed genes tested and this association correlates with binding of H3K4me2. Thus, our study has identified a novel H3K4me2 binding protein and revealed a role of NRDc in transcriptional regulation.

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Lipid polarity is maintained in the absence of tight junctions.

Publication Date: 2012 Jan 31 PMID: 22294698
Authors: Ikenouchi, J. - Suzuki, M. - Umeda, K. - Ikeda, K. - Taguchi, R. - Kobayashi, T. - Sato, S. B. - Kobayashi, T. - Stolz, D. B. - Umeda, M.
Journal: J Biol Chem

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with the fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. Our present study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.

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The ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity.

Publication Date: 2012 Jan 31 PMID: 22294697
Authors: Ulrich, D. L. - Lynch, J. - Wang, Y. - Fukuda, Y. - Nachagari, D. - Du, G. - Sun, D. - Fan, Y. - Tsurkan, L. - Potter, P. M. - Rehg, J. E. - Schuetz, J.
Journal: J Biol Chem

Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient ( (Abcb6+/-) ) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, ( Abcb6-/- ) mice derived from these stem cells appeared phenotypically normal. We hypothesized other ATP-dependent and/or independent mechanisms conserve porphyrins. Here, we demonstrate that ( Abcb6-/- ) mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in upregulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in ( Abcb6-/- ) mice, possibly due to sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer and loss of Abcb6 produces upregulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g., phenylhydrazine stress), these adaptations appear inadequate which suggests, under these conditions Abcb6 is important for optimal survival.

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Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damage.

Publication Date: 2012 Jan 31 PMID: 22294696
Authors: Paget, C. - Ivanov, S. - Fontaine, J. - Renneson, J. - Blanc, F. - Pichavant, M. - Dumoutier, L. - Ryffel, B. - Renauld, J. C. - Gosset, P. - Gosset, P. - Si-Tahar, M. - Faveeuw, C. - Trottein, F.
Journal: J Biol Chem

Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive ab T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here, we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-g and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells whilst the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1b and IL-23 provided by infected DCs, independently of the CD1d molecule, to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.

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Human primary keratinocytes show a restricted ability to up-regulate suppressor of cytokine signaling (SOCS)3 compared to autologous macrophages.

Publication Date: 2012 Jan 31 PMID: 22294695
Authors: Zeitvogel, J. - Dalpke, A. - Eiz-Vesper, B. - Kracht, M. - Dittrich-Breiholz, O. - Werfel, T. - Wittmann, M.
Journal: J Biol Chem

Suppressor of Cytokine Signaling (SOCS)3 belongs to a family of proteins that are known to exert important functions as inducible feedback inhibitors and are crucial for the balance of immune responses. There is evidence for a de-regulated immune response in chronic inflammatory skin diseases. Thus it was the aim of this study to investigate the regulation of SOCS proteins involved in intracellular signaling pathways occurring during inflammatory skin diseases and analyze their impact on the course of inflammatory responses. As we and others have previously described that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases, we focused here on the signaling induced by IL-27 in human primary keratinocytes as compared with autologous blood derived macrophages. Here, we demonstrate that SOCS3 is critically involved in regulating the cell-specific response to IL-27. SOCS3 was found to be significantly up-regulated by IL-27 in macrophages but not in keratinocytes. Other STAT3 activating cytokines investigated including IL-6, IL-22 or OSM also failed to up-regulate SOCS3 in keratinocytes. Lack of SOCS3 up-regulation in skin epithelial cells was accompanied by prolonged STAT1 and STAT3 phosphorylation and enhanced CXCL10 production upon IL-27 stimulation as compared with macrophages. Overexpression of SOCS3 in keratinocytes significantly diminished this enhanced CXCL10 production in response to IL-27. We conclude from our data that keratinocytes have a cell-type specific impaired capacity to upregulate SOCS3 which may crucially determine the course of chronic inflammatory skin diseases.

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Crystal structure of a novel metallo-carboxypeptidase inhibitor from the marine mollusk Nerita versicolor in complex with human carboxypeptidase A4.

Publication Date: 2012 Jan 31 PMID: 22294694
Authors: Covaleda, G. - Alonso Del Rivero, M. - Chavez, M. A. - Aviles, F. X. - Reverter, D.
Journal: J Biol Chem

NvCI is a novel exogenous proteinaceous inhibitor of metallo-carboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase 4 (hCPA4) and NvCI has been crystallized and determined at 1.7 A resolution. The NvCI structure defines a distinctive protein fold basically composed by a two-stranded antiparallel beta-sheet connected by three loops, the inhibitory C-terminal tail and stabilized by three disulphide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with hCPA4, responsible of inhibitory constant values in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.

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Structural and functional characterization of BC28.1, the major erythrocyte binding protein from Babesia canis merozoite surface.

Publication Date: 2012 Jan 31 PMID: 22294693
Authors: Yang, Y. S. - Murciano, B. - Moubri, K. - Cibrelus, P. - Schetters, T. - Gorenflot, A. - Delbecq, S. - Roumestand, C.
Journal: J Biol Chem

Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of protozoa parasites from the genus Babesia. Like Plasmodium falciparum, the agent of Malaria, or Toxoplasma gondii, responsible for the human toxoplasmosis, Babesia belongs to the Apicomplexa family. Babesia canis is the agent of the canine babesiosis in Europe. The identification and characterisation of parasite surface proteins represent major goals, both for the understanding of the Apicomplexa invasion process and for their vaccine potential. Indeed, we have already shown that Bd37, the major antigenic adhesion protein from Babesia divergens, was able to induce complete protection against various parasite strains. The major merozoite surface antigens of Babesia canis have been described as a 28 kDa membrane protein family, anchored at the surface of the merozoite. Here, we demonstrate that Bc28.1, a major member of this multigenic family, is expressed at high levels at the surface of the merozoite. We defined the erythrocyte binding function of Bc28.1 and determined its high-resolution solution structure using NMR spectroscopy. Surprisingly, although these proteins are thought to play a similar role in the adhesion process, the structure of Bc28.1 from Babesia canis appears unrelated to the previously published structure of Bd37 from Babesia divergens. Site-directed mutagenesis experiments also suggest that the mechanism of the interaction with erythrocyte membrane could be different for the two proteins. The resolution of the structure of Bc28 represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.

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Increasing serum half-life and extending cholesterol lowering in vivo by engineering an antibody with pH-sensitive binding to PCSK9.

Publication Date: 2012 Jan 31 PMID: 22294692
Authors: Chaparro-Riggers, J. - Liang, H. - Devay, R. M. - Bai, L. - Sutton, J. E. - Chen, W. - Geng, T. - Lindquist, K. - Galindo Casas, M. - Boustany, L. M. - Brown, C. L. - Chabot, J. - Gomes, B. - Garzone, P. - Rossi, A. - Strop, P. - Shelton, D. - Pons, J. - Rajpal, A.
Journal: J Biol Chem

Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus and human cross-reactive, antagonistic anti-Proprotein Convertase Substilisin Kexin type 9 (PCSK9) antibodies J10 and the affinity matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic (PK) profiles. These antibodies prevent the degradation of Low Density Lipoprotein receptor (LDLR) thus lowering serum levels of LDL-cholesterol (LDL-C) and potently reducing serum cholesterol in mice and selectively reduce LDL-C in cynomolgus monkeys. In order to increase the PK and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increase duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9, in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, while the antibody-antigen complex dissociates at the endosomal pH of around 5 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increasing the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor (FcRn), which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.

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MicroRNA profile of circulating CD4-positive regulatory T cells in human adults and the impact of differentially expressed microRNAs on the expression of two genes essential to their function.

Publication Date: 2012 Jan 31 PMID: 22294691
Authors: Fayyad-Kazan, H. - Rouas, R. - Fayyad-Kazan, M. - Badran, R. - El Zein, N. - Lewalle, P. - Najar, M. - Hamade, E. - Jebbawi, F. - Merimi, M. - Romero, P. - Burny, A. - Badran, B. - Martiat, P.
Journal: J Biol Chem

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor alpha chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4+ Tregs isolated from adult healthy donors, and identified a signature composed of fifteen differentially expressed microRNAs. Among those, miR-24, miR-145 and miR-210 were down regulated in Tregs compared to controls and were found to have potential target sites in the 3 UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3 UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4+ adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3 UTR. To our knowledge, this is the first identification of human adult peripheral blood CD4+ Tregs microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

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Mutated MHC class II transactivator upregulates IL-33-dependent differentiation of Th2 subset through Nod2 binding for NLR signaling initiation.

Publication Date: 2012 Jan 31 PMID: 22294690
Authors: Yang, Z. - Gu, P. - Shao, X. - Li, Z. - Zhou, Y. - Lu, L. - Chou, K. Y.
Journal: J Biol Chem

Dominant-negative mutants of class II transactivator (mCIITAs) with N-terminus depletion have been used to repress the transcription of class II genes in xenotransplantation. Here, we report that mCIITA overexpressing myeloid cell line Ana-1 (Ana-1-mCIITA) derived from a C57BL/6 mouse was able to down-regulate the MHC class II expression and reverse immune responses from Th1 (IL-2(+) IFN-gamma(+)STAT4(+)) to Th2 (IL-4(+)IL-5(+)IL-10(+)IL-13(+)STAT6(+)) when cocultured with T cells. Mechanism analysis indicated that the mCIITA protein is able to initiate a NOD-like receptor (NLR)-related signaling pathway via binding of the cytoplasmic Nod2 protein, which was followed by activating RIP2, caspase 1 and IKK-alpha/beta. This ensures the expression of the genes encoding the cytokines IL-33, IL-1beta and TNF-alpha: however, only the highly expressed IL-33 is responsible for inducing the type 2 response, with a skewed Th2 cytokine secretion (IL-4(+)IL-5(+)IL-10(+)IL-13(+)IL-2(-)IFN-gamma(-)), which was completely prevented by the deactivation of the Nod2 gene with siRNA or by the blockage of the IL-33-related signaling using the mAb ST2L against the IL-33 receptor. mCIITA-mediated Th2 conversion was also successfully induced in vivo in a mCIITA-transgenic C57BL/6 mouse model. These results indicate that the Th1/Th2 balance could be regulated by an N-terminus-depleted CIITA molecule via NLR-related signaling, a property valuable for disease control, especially for inducing transplantation tolerance via the repression of class II expression and the attenuation of a Th1-dominant response.

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Genome-wide repression of NF-kappaB target genes by the transcription factor MIBP1 and its modulation by O-GlcNAc transferase.

Publication Date: 2012 Jan 31 PMID: 22294689
Authors: Iwashita, Y. - Fukuchi, N. - Waki, M. - Hayashi, K. - Tahira, T.
Journal: J Biol Chem

The transcription factor c-MYC intron binding protein 1 (MIBP1) binds to various genomic regulatory regions, including intron 1 of c-MYC. This factor is highly expressed in post-mitotic neurons in the fetal brain and may be involved in various biological steps, such as neurological and immunological processes. In this study, we globally characterized the transcriptional targets of MIBP1 and proteins that interact with MIBP1. Microarray hybridization followed by Gene Set Enrichment Analysis revealed that genes involved in the pathways downstream of MYC, NF-kappaB, and TGF-beta were downregulated when HEK293 cells stably overexpressed MIBP1. In silico transcription factor binding site analysis of the promoter regions of these downregulated genes showed that the NF-kappaB binding site was the most overrepresented. The upregulation of genes known to be in the NF-kappaB pathway after the knockdown of endogenous MIBP1 in HT1080 cells supports the view that MIBP1 is a downregulator of the NF-kappaB pathway. We also confirmed the binding of the MIBP1 to the NF-kappaB site. By immunoprecipitation and mass spectrometry, we detected O-linked beta-N-acetylglucosamine (O-GlcNAc) transferase (OGT) as a prominent binding partner of MIBP1. Analyses using deletion mutants revealed that a 154-amino acid region of MIBP1 was necessary for its OGT binding and O-GlcNAcylation. A luciferase reporter assay showed that NF-kappaB-responsive expression was repressed by MIBP1, and stronger repression by MIBP1 lacking the 154-amino acid region was observed. Our results indicate that the primary effect of MIBP1 expression is the downregulation of the NF-kappaB pathway, and that this effect is attenuated by O-GlcNAc signaling.

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G-protein-coupled receptor kinase interactor-1 (GIT-1) is a new endothelial nitric oxide synthase (eNOS) interactor with functional effects on vascular homeostasis.

Publication Date: 2012 Jan 31 PMID: 22294688
Authors: Liu, S. - Premont, R. T. - Rockey, D. C.
Journal: J Biol Chem

Endothelial cell nitric oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in the vasculature, undergoes extensive post-translational modifications that modulate its activity. Here we have identified a novel eNOS interactor, G-protein-coupled receptor kinase interactor-1 (GIT1), which plays an unexpected role in G-protein-coupled receptor (GPCR)-stimulated NO signaling. GIT1 interacted with eNOS in the endothelial cell cytoplasm, and this robust association was associated with stimulatory eNOS phosphorylation (Ser1177), enzyme activation, and NO synthesis. GIT1 knockdown had the opposite effect. Additionally, GIT1 expression was reduced in sinusoidal endothelial cells after liver injury, consistent with previously described endothelial dysfunction in this disease. Re-expression of GIT1 after liver injury rescued the endothelial phenotype. These data emphasize the role of G-protein-coupled receptor (GPCR) signaling partners in eNOS function and have fundamental implications for vascular disorders involving dysregulated eNOS.

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Crystal structures and small-angle X-ray scattering analysis of UDP-galactopyranose mutase from the pathogenic fungus Aspergillus fumigatus.

Publication Date: 2012 Jan 31 PMID: 22294687
Authors: Dhatwalia, R. - Singh, H. - Oppenheimer, M. - Karr, D. B. - Nix, J. C. - Sobrado, P. - Tanner, J. J.
Journal: J Biol Chem

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans makes UGM a potential drug target. Here we report crystal structures and small-angle X-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle X-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 A to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.

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Human alpha/beta hydrolase domain containing 10 (ABHD10) is a responsible enzyme for deglucuronidation of mycophenolic acid acyl-glucuronide in liver.

Publication Date: 2012 Jan 31 PMID: 22294686
Authors: Iwamura, A. - Fukami, T. - Higuchi, R. - Nakajima, M. - Yokoi, T.
Journal: J Biol Chem

Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is primarily metabolized by glucuronidation to a phenolic glucuronide (MPAG) and an acyl glucuronide (AcMPAG). It is known that AcMPAG, which may be an immunotoxic metabolite, is deglucuronidated in human liver. However, it has been reported that recombinant beta-glucuronidase does not catalyze this reaction. AcMPAG deglucuronidation activity was detected in both human liver cytosol (HLC) and microsomes (HLM). In this study, the enzyme responsible for AcMPAG deglucuronidation was identified by purification from HLC with column chromatographic purification steps. The purified enzyme was identified as alpha/beta hydrolase domain containing 10 (ABHD10) by amino acid sequence analysis. Recombinant ABHD10 expressed in Sf9 cells efficiently deglucuronidated AcMPAG with a Km value of 100.7 +/- 10.2 microM, which was similar to those in HLM, HLC, and human liver homogenates (HLH). Immunoblot analysis revealed ABHD10 protein expression in both HLC and HLM. The AcMPAG deglucuronidation by recombinant ABHD10, HLC, and HLH were potently inhibited by AgNO3, CdCl2, CuCl2, PMSF, BNPP, and DTNB. The CLint value of AcMPAG formation from MPA, which was catalyzed by human UGT2B7, in HLH was increased by 1.8-fold in the presence of PMSF. Thus, human ABHD10 would affect the formation of AcMPAG, the immunotoxic metabolite.

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Nurr1 is required for NMDA receptor-mediated neuronal survival.

Publication Date: 2012 Jan 31 PMID: 22294685
Authors: Barneda-Zahonero, B. - Servitja, J. M. - Badiola, N. - Minano-Molina, A. J. - Fado, R. - Saura, C. A. - Rodriguez-Alvarez, J.
Journal: J Biol Chem

NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and maturate during its migration from the external granular layer (EGL) to the internal granular layer (IGL) and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5mM) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole-genome expression profiles after 3 and 8 hours of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR sitimulation. ChiP assay confirmed CREB bindig to Nurr1 promoter whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Morevoer, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in BDNF protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons.

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THE PROTEOME OF THE HYDRA NEMATOCYST.

Publication Date: 2012 Jan 30 PMID: 22291027
Authors: Balasubramanian, P. G. - Beckmann, A. - Warnken, U. - Schnoelzer, M. - Schueler, A. - Bornberg-Bauer, E. - Holstein, T. W. - Ozbek, S.
Journal: J Biol Chem

Stinging cells or nematocytes of jellyfish and other cnidarians represent one of the most poisonous and sophisticated cellular inventions in animal evolution. This ancient cell type is unique in containing a giant secretory vesicle derived from the Golgi apparatus. The organelle structure within the vesicle comprises an elastically stretched capsule (nematocyst) to which a long tubule is attached. During exocytosis the barbed part of the tubule is accelerated with > 5 million g in less than 700 ns enabling a harpoon-like discharge. Hitherto the molecular components responsible for the organelles biomechanical properties were largely unknown. Here, we describe the proteome of nematocysts from the freshwater polyp Hydra magnipapillata. Our analysis revealed an unexpectedly complex secretome of 410 proteins with venomous and lytic, but also adhesive or fibrous properties. In particular, the insoluble fraction of the nematocyst represents a functional extracellular matrix (ECM) structure of collagenous and elastic nature. This finding suggests an evolutionary scenario in which exocytic vesicles harboring a venomous secretome assembled a sophisticated predatory structure from ECM motif proteins.

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On the potential interactions between Non-Selective cation channel TRPM4 and the sulfonylurea receptor SUR1.

Publication Date: 2012 Feb 1 PMID: 22291026
Authors: Sala-Rabanal, M. - Wang, S. - Nichols, C. G.
Journal: J Biol Chem

The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the Transient Receptor Potential family of non-selective cation (NSC) channels. It has been reported that, following stroke, brain or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by upregulation of a NSC conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic NSC currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP, but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional KATP channels were formed. In cells cotransfected with Kir6.2, SUR1 and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity, but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, while there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.

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Functional homomers and heteromers of dopamine D2L and D3 receptors co-exist at the cell surface.

Publication Date: 2012 Jan 30 PMID: 22291025
Authors: Pou, C. - Mannoury la Cour, C. - Stoddart, L. A. - Millan, M. J. - Milligan, G.
Journal: J Biol Chem

Human dopamine D(2long) and D(3) receptors were modified by N-terminal addition of SNAP or CLIP forms of O6-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP-and CLIP-tag covalent labelling reagents and, based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D(2long) and D(3) receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D(2long)-D(3) heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D(2long)-D(3) heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D(2long) and D(3) receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D(2long) and D(3) receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady-state. These data are of potential importance both to disorders in which D(2long) and D(3) receptors are implicated, like schizophrenia and Parkinsons disease, and also to drugs exerting their actions via these sites.

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Small GTPase Rab17 regulates the dendritic morphogenesis and postsynaptic development of hippocampal neurons.

Publication Date: 2012 Jan 30 PMID: 22291024
Authors: Mori, Y. - Matsui, T. - Furutani, Y. - Yoshihara, Y. - Fukuda, M.
Journal: J Biol Chem

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains, axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases, which are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

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Short-chain dehydrogenase/reductase rdhe2 is a novel retinol dehydrogenase essential for frog embryonic development.

Publication Date: 2012 Jan 30 PMID: 22291023
Authors: Belyaeva, O. V. - Lee, S. A. - Adams, M. K. - Chang, C. - Kedishvili, N. Y.
Journal: J Biol Chem

The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short-chain dehydrogenase/ reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates.

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Mutations in the N-terminal flanking region of the blue-light sensing domain LOV2 disrupt its repressive activity on the kinase domain in the Chlamydomonas phototropin.

Publication Date: 2012 Jan 30 PMID: 22291022
Authors: Aihara, Y. - Yamamoto, T. - Okajima, K. - Yamamoto, K. - Suzuki, T. - Tokutomi, S. - Tanaka, K. - Nagatani, A.
Journal: J Biol Chem

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning and stomata opening in plants to increase the photosynthetic efficiency. Blue-light stimulus first induces local conformational changes in the chromophore-bearing Light-Oxygen and Voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C-terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue-light dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We then applied this system to a large-scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jalpha region, appears to play a crucial role in the regulation of kinase activity in phototropin.

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S1P-lyase-deficiency increases sphingolipid formation via recycling at the expense of de novo biosynthesis in neurons.

Publication Date: 2012 Jan 30 PMID: 22291021
Authors: Hagen-Euteneuer, N. - Luetjohann, D. - Park, H. - Merrill, A. H. Jr - van Echten-Deckert, G.
Journal: J Biol Chem

Sphingosine-1-phosphate-lyase (S1P-lyase) irreversibly cleaves sphingosine-1-phosphate (S1P) in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphate are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. Indeed, we have previously reported that S1P-lyase-deficiency causes neurodegeneration and other adverse symptoms. We next asked the question whether and how S1P-lyase-deficiency affects the metabolism of (glyco)sphingolipids and cholesterol, two lipid classes that might be involved in the neurodegenerative processes observed in S1P-lyase-deficient mice. As predicted, there was a considerable increase in free and phosphorylated sphingoid bases upon elimination of S1P-lyase, but to our surprise, rather than increase, the mass of (glyco)sphingolipids persisted at wild type levels. This was discovered to be due to reduced de novo sphingoid base biosynthesis and a corresponding increase in the recycling of the backbones via the salvage pathway. There was also a considerable increase in cholesterol esters, although free cholesterol persisted at wild type levels, which might be secondary to the shifts in sphingolipid metabolism. All in all, these findings show that accumulation of free and phosphorylated sphingoid bases by loss of S1P-lyase causes an interesting readjustment of the balance between de novo biosynthesis and recycling to maintain (glyco)sphingolipid homeostasis. These changes, and their impact on the metabolism of other cellular lipids, should be explored as possible contributors to the neurodegeneration in S1P-lyase-deficiency.

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5-Hydroxytryptamine 5HT2C receptors form a protein complex with N-methyl-D-Aspartate GLUN2A subunits and activate phosphorylation of Src to modulate motoneuronal depolarization.

Publication Date: 2012 Jan 30 PMID: 22291020
Authors: Bigford, G. E. - Chaundry, N. S. - Keane, R. W. - Holohean, A. M.
Journal: J Biol Chem

NMDA-gated ion channels are known to play a critical role in motoneuron depolarization, but the molecular mechanisms modulating NMDA activation in the spinal cord are not well understood. Our present work demonstrates that activated 5HT2C receptors enhance NMDA depolarizations recorded electrophysiologically from motoneurons. Pharmacological studies indicate involvement of src tyrosine kinase mediates 5HT2C-facilitation of NMDA. Rt-PCR analysis revealed edited forms of 5HT2C were present in mammalian spinal cord, indicating the availability of G-protein-independent isoforms. Spinal cord neurons treated with the 5HT2C agonist MK 212 showed increased srcTyr416 phosphorylation in a dose-dependent manner thus verifying that src is activated after treatment. In addition, 5HT2C antagonists and tyrosine kinase inhibitors blocked 5HT2C-mediated srcTyr416 phosphorylation and also enhanced NMDA-induced motoneuron depolarization. Co-immunoprecipitation of synaptosomal fractions showed that GluN2A, 5HT2C receptors and src tyrosine kinase form protein associations in synaptosomes. Moreover, immunohistochem-ical analysis demonstrated GluN2A and 5HT2C receptors co-localize on the processes of spinal neurons. These findings reveal that a distinct multi-protein complex links 5HT-activated intracellular signaling events with NMDA-mediated functional activity.

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An unique iron coordination in the iron-chelating molecule vibriobactin helps Vibrio cholera evade the mammalian siderocalin-mediated immune response.

Publication Date: 2012 Jan 30 PMID: 22291019
Authors: Li, N. - Zhang, C. - Li, B. - Liu, X. - Huang, Y. - Xu, S. - Gu, L.
Journal: J Biol Chem

Iron is essential for the survival of almost all bacteria. Vibrio cholera acquires iron through the secretion of a catecholate siderophore called vibriobactin. At present, how vibriobactin chelates ferric ion remains controversial. In addition, the mechanisms underlying the recognition of ferric-vibriobactin by the siderophore transport system and its delivery into the cytoplasm specifically have not been clarified. In this study, we report high-resolution structures of the ferric-vibriobactin periplasmic binding protein ViuP and its complex with ferric-vibriobactin. The holo ViuP structure revealed that ferric-vibriobactin does not adopt the same iron coordination as that of other catecholate siderophores such as enterobactin. The three catechol moieties donate five, rather than six, oxygen atoms as iron ligands. The sixth iron ligand is provided by a nitrogen atom from the second oxazoline ring. This kind of iron coordination results in the protrusion of the second catechol moiety and renders the electrostatic surface potential of ferric vibriobactin less negatively polarized compared with ferric-enterobactin. To accommodate ferric-vibriobactin, ViuP has a deeper subpocket to hold the protrusion of the second catechol group. This structural characteristic has not been observed in other catecholate siderophore binding proteins. Biochemical data showed that siderocalin, which is part of the mammalian innate immune system, cannot efficiently sequester ferric-vibriobactin in vitro although it can capture many catecholate siderophores with high efficiency. Our findings suggest that the unique iron coordination found in ferric-vibriobactin may be utilized by some pathogenic bacteria to evade siderocalin-mediated innate immune response of mammals.

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Mapping of the localization of type I angiotensin receptor in membrane microdomains using bioluminescence resonance energy transfer-based sensors.

Publication Date: 2012 Jan 30 PMID: 22291018
Authors: Balla, A. - Toth, D. - Soltesz-Katona, E. - Szakadati, G. - Erdelyi, L. S. - Varnai, P. - Hunyady, L.
Journal: J Biol Chem

Initiation and termination of signaling of the type I angiotensin receptor (AT1-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft-, or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT1-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer (BRET) measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY/AAY or TSTS/A) AT1-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT1-R upon AngII stimulus to those of [Sar1,Ile8]-AngII or [Sar1,Ile4,Ile8]-AngII stimulus revealed different type of changes depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT1-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the BRET-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments.

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The adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins Ezrin/Radixin/Moesin by Mammalian Ste20-4 to protect cells from oxidative stress.

Publication Date: 2012 Jan 30 PMID: 22291017
Authors: Fidalgo, M. - Guerrero, A. - Fraile, M. - Iglesias, C. - Pombo, C. M. - Zalvide, J.
Journal: J Biol Chem

While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress which results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.

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tRNA controlled nuclear import of a human tRNA synthetase.

Publication Date: 2012 Jan 30 PMID: 22291016
Authors: Fu, G. - Xu, T. - Shi, Y. - Wei, N. - Yang, X. L.
Journal: J Biol Chem

Aminoacyl-tRNA synthetases, essential components of the cytoplasmic translation apparatus, also have nuclear functions that continue to be elucidated. However, little is known about how the distribution between cytoplasmic and nuclear compartments is controlled. Using a combination of methods, here we showed that human tyrosyl-tRNA synthetase (TyrRS) distributes to the nucleus and that the nuclear import of human TyrRS is regulated by its cognate tRNATyr. We identified a hexapeptide motif in the anticodon recognition domain that is critical for nuclear import of the synthetase. Remarkably, this nuclear localization sequence (NLS) motif is also important for interacting with tRNATyr. As a consequence, mutational alteration of the hexapeptide simultaneously attenuated aminoacylation and nuclear localization. Because the NLS is sterically blocked when the cognate tRNA is bound to TyrRS, we hypothesized that the nuclear distribution of TyrRS is regulated by tRNATyr. This expectation was confirmed by RNAi-knockdown of tRNATyr expression, which led to robust nuclear import of TyrRS. Further bioinformatics analysis showed that, to have nuclear import of TyrRS directly controlled by tRNATyr in higher organisms, the NLS of lower eukaryotes was abandoned while the new NLS was evolved from an anticodon-binding hexapeptide motif. Thus, higher organisms developed a strategy to make tRNA a regulator of the nuclear trafficking of its cognate synthetase. The design in principle should coordinate nuclear import of a tRNA synthetase with the demand of protein synthesis in the cytoplasm.

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Using Intramolecular Disulfide Bonds in Tau Protein to Deduce Structural Features of Aggregation-resistant Conformations.

Publication Date: 2012 Jan 30 PMID: 22291015
Authors: Walker, S. - Ullman, O. - Stultz, C. M.
Journal: J Biol Chem

As tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence tau's aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, while there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. Although it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are little data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, that contains all 4 microtubule binding repeats, and isolate the monomeric fraction, corresponding to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-beta structure. These data further our understanding of the precise structural features that retard tau aggregation.

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Structural and biochemical characterization of human mitochondrial branched-chain alpha-ketoacid dehydrogenase phosphatase.

Publication Date: 2012 Jan 30 PMID: 22291014
Authors: Wynn, R. M. - Li, J. - Brautigam, C. A. - Chuang, J. L. - Chuang, D. T.
Journal: J Biol Chem

The branched-chain alpha-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn2+ ions for phosphatase activity, whereas Mg2+ and Ca2+ ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nM and 10 muM for Mn2+ and Mg2+ ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca2+-independent binding of BDP to the 24-meric transacylase (E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by X-ray crystallography to 2.4-A resolution. The BDP structure is dominated by a central beta-sandwich. There are two protrusions forming a narrow cleft of ~10-A wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp109, Asp207, Asp298 and Asp337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg104 increases the Km for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

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NOX5 in human spermatozoa: Expression, function and regulation.

Publication Date: 2012 Jan 30 PMID: 22291013
Authors: Musset, B. - Clark, R. A. - Decoursey, T. E. - Petheo, G. L. - Geiszt, M. - Chen, Y. - Cornell, J. E. - Eddy, C. A. - Brzyski, R. G. - El Jamali, A.
Journal: J Biol Chem

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+ )chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous over-expression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moroever, the HV1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.

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Lung cancer-derived galectin-1 enhances tumorigenic potentiation of tumor associated dendritic cells by expressing HB-EGF.

Publication Date: 2012 Jan 30 PMID: 22291012
Authors: Kuo, P. L. - Huang, M. S. - Cheng, D. E. - Hung, J. Y. - Yang, C. J. - Chou, S. H.
Journal: J Biol Chem

The interaction between cancer cells and their microenvironment is a vicious cycle that enhances the survival and progression of cancer, resulting in metastasis. This study is the first to indicate that lung cancer-derived galectin-1 secretion is responsible for stimulating tumor associated dendritic cells (TADCs) production of mature heparin-binding EGF-like growth factor (HB-EGF), which in turn increases cancer progression. Treatment of galectin-1, present in large amounts in lung cancer condition medium (CM) and lung cancer patient sera, mimicked the inductive effect of lung cancer-CM on the expression and ectodomain shedding of HB-EGF by TNFalpha-converting enzyme/a disintegrin and metalloproteinase 9 (ADAM9) and ADAM17. Significant upregulation of HB-EGF has been seen in tumor-infiltrating CD11c+ DCs in human lung cancer samples. Active cleavage of HB-EGF in TADCs by ADAM9 and ADAM17 is associated with increased protein kinase C delta and Lyn signaling. Enhancement of HB-EGF production in TADCs increased lung cancer proliferation, migration and epithelial-to-mesenchymal transition abilities. In contrast, inhibiting HB-EGF by siRNA suppressed TADC-mediated cancer progression. Moreover, mice injected with galectin-1 knockdown Lewis lung carcinoma showed decreased expression and ectodomain shedding of HB-EGF and reduced incidence of cancer development, resulting in increased survival rates. We demonstrate here for the first time that human and mouse DCs are a source of HB-EGF, an EGFR ligand with tumorigenic properties. Antagonists of the effect of lung cancer-derived galectin-1 effect on DCs and anti-HB-EGF blocking antibodies could therefore have therapeutic potential as antitumor agents.

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Insights into the structural network responsible for oligomerization and activity Of the bacterial virulence regulator caseinolytic protease P (ClpP).

Publication Date: 2012 Jan 30 PMID: 22291011
Authors: Gersch, M. - List, A. - Groll, M. - Sieber, S. A.
Journal: J Biol Chem

The barrel-shaped ClpP protease is a main virulence regulator in the bacterial pathogen Staphylococcus aureus. It consists of two heptameric rings forming a homo-tetradecamer with an inner chamber that houses the 14 active sites. We recently showed that SaClpP3 is able to adopt a compressed, inactive conformation. We here present the 2.3 A resolution structure of SaClpP in its closed, active conformation as well as the structure of the S98A mutant. Comprehensive mutational analysis aiming at destabilizing one or the other or both conformations was able to pinpoint key residues involved in this catalytic switch and in the heptamer-heptamer interaction. By probing the active site serine with a covalently modifying beta-lacton probe, we could show that the tetradecameric organization is essential for a proper formation of the active site. Structural data suggest that a highly conserved hydrogen-bonding network links oligomerization to activity. A comparison of ClpP structures from different organisms provides suggestive evidence for the presence of a universal mechanism regulating ClpP protease activity in which binding of one subunit to the corresponding subunit on the other ring interface is necessary for the functional assembly of the catalytic triad and thus for protease function. This mechanism ensures controlled access to the active sites of a highly unspecific protease.

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4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter.

Publication Date: 2012 Jan 30 PMID: 22291010
Authors: Solis, E. - Zdravkovic, I. - Tomlinson, I. D. - Noskov, S. Y. - Rosenthal, S. J. - De Felice, L. J.
Journal: J Biol Chem

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now their use has mostly been successful to study the catecholamine transporters, but not the serotonin (5HT) transporter. Here we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents, and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand-receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.

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A murine model of dextran sulfate sodium-induced colitis reveals Candida glabrata virulence and contribution of beta-Mannosyltransferases.

Publication Date: 2012 Jan 30 PMID: 22291009
Authors: Jawhara, S. - Mogensen, E. - Maggiotto, F. - Fradin, C. - Sarazin, A. - Dubuquoy, L. - Maes, E. - Guerardel, Y. - Janbon, G. - Poulain, D.
Journal: J Biol Chem

Candida glabrata, like Candida albicans, is an opportunistic yeast pathogen that has adapted to colonize all segments of the human gastrointestinal tract and vagina. The C. albicans cell wall expresses beta-1,2-linked mannosides (beta-Mans) promoting its adherence to host cells and tissues. As beta-Mans are also present in C. glabrata, their role in C. glabrata colonization and virulence was investigated in a murine model of dextran sulfate sodium (DSS)-induced colitis. Five clustered genes of C. glabrata encoding beta-mannosyltransferases, BMT2-BMT6, were deleted simultaneously. beta-Man expression was studied by Western blotting, flow cytometry and NMR analysis. Mortality, clinical, histologic, and colonization scores were determined in mice receiving DSS and different C. glabrata strains. The results show that C. glabrata bmt2-6 strains had a significant reduction in beta-1,2 Man expression and a disappearance of beta-1,2 mannobiose in the acid-stable domain. A single gavage of C. glabrata wild-type strain in mice with DSS-induced colitis caused a loss of body weight, colonic inflammation, and mortality. Mice receiving C. glabrata bmt2-6 mutant strains had normal body weight and reduced colonic inflammation. Lower numbers of colonies of C. glabrata bmt2-6 were recovered from stools and different parts of the gastrointestinal tract. Histopathologic examination revealed that the wild-type strain had a greater ability to colonize tissue and cause tissue damage. These results showed that C. glabrata has a high pathogenic potential in DSS-induced colitis where beta-Mans contribute to colonization and virulence.

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Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors.

Publication Date: 2012 Jan 30 PMID: 22291008
Authors: Comas-Casellas, E. - Martinez-Barriocanal, A. - Miro, F. - Ejarque-Ortiz, A. - Schwartz, S. - Martin, M. - Sayos, J.
Journal: J Biol Chem

Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human PBMC and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking of any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with all the CD300 family members, with the exception of CD300c. Contrary to other activating members of CD300 family of receptors, surface expression of CD300d in COS-7 transfected cells required the presence of an ITAM-bearing adaptor (FcepsilonRgamma). Accordingly, we found that CD300d was able to recruit FcepsilonRgamma. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcepsilonRgamma, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules did not modify either the intracellular expression of CD300d. In fact, the presence of CD300d decreases the levels of surface expression of CD300f, but not CD300c. Our data suggests that the function of CD300d would be related with the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.

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Mechanisms of host receptor adaptation by SARS coronavirus.

Publication Date: 2012 Jan 30 PMID: 22291007
Authors: Wu, K. - Peng, G. - Wilken, M. - Geraghty, R. J. - Li, F.
Journal: J Biol Chem

SARS coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors have been poorly understood, presenting evolutionary and epidemic conundrums. This study investigates the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we have elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hotspots on ACE2, and by doing so they enhance viral interactions with either human or civet ACE2. Therefore, these mutations were viral adaptations to either human or civet ACE2. To corroborate the above analysis, we have designed and characterized two optimized RBDs. The human-optimized RBD contains all of the human-ACE2-adapted residues (F442/F472/N479/D480/T487), and possesses exceptionally high affinity for human ACE2 but relative low affinity for civet ACE2. The civet-optimized RBD contains all of the civet-ACE2-adapted residues (P442/Y472/R479/G480/T487), and possesses exceptionally high affinity for civet ACE2 and also substantial affinity for human ACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV, but also provide molecular and structural basis for tracking future SARS-CoV evolution in animals.

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Studying Reactive Species in a Single Shot{diamondsuit}: Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems. High-throughput Real-time Analyses.

Publication Date: 2012 Jan 27 PMID: 22287685
Authors:
Journal: J Biol Chem

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An oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from the formation of reactive oxygen species.

Publication Date: 2012 Jan 27 PMID: 22287551
Authors: Paulus, A. - Rossius, S. G. - Dijk, M. - de Vries, S.
Journal: J Biol Chem

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low-spin heme b558 that donates electrons to a binuclear heme b595 / heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-Vis spectroscopy. After the initial binding of O2, the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin pi-cation radical intermediate (Compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75 - 0.85 per enzyme in agreement with its much higher rate of formation (~ 20,000 s-1) compared to its rate of decay (~ 1,900 s-1). Compound I is next converted to a short-lived heme d oxoferryl intermediate (Compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of Reactive Oxygen Species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of Reactive Oxygen Species.

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NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes.

Publication Date: 2012 Jan 27 PMID: 22287546
Authors: Han, C. Y. - Umemoto, T. - Omer, M. - Den Hartigh, L. J. - Chiba, T. - Leboeuf, R. - Buller, C. L. - Sweet, I. R. - Pennathur, S. - Abel, E. D. - Chait, A.
Journal: J Biol Chem

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250muM) in either 5mM or 25mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX 4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX and pentose phosphate pathway (PPP) activity, increased with glucose but not palmitate, and decreased with DHA exposure. Inhibition of PPP by glucose-6 phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.

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Insights into hemoglobin assembly through in vivo mutagenesis of alpha-hemoglobin stabilizing protein.

Publication Date: 2012 Jan 27 PMID: 22287545
Authors: Khandros, E. - Mollan, T. L. - Yu, X. - Wang, X. - Yao, Y. - D'Souza, J. - Gell, D. A. - Olson, J. S. - Weiss, M. J.
Journal: J Biol Chem

Alpha-Hemoglobin Stabilizing Protein (AHSP) is believed to facilitate adult Hemoglobin A (HbA) assembly and protect against toxic free alpha globin subunits. Recombinant AHSP binds multiple forms of free alpha globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of alphaO2 subunit and its rapid conversion to a partially unfolded bis-histidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant binds oxygenated alpha chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also cause decreased rates of alphaO2 autooxidation as compared to wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibit no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions have minimal effects on AHSP interactions with ferric alpha subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent alpha chain folding intermediates prior to hemin reduction and incorporation into HbA is physiologically more important than AHSP interactions with ferrous alphaO2 subunits.

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Regulation of NMDA receptors by metabotropic glutamate receptor 7.

Publication Date: 2012 Jan 27 PMID: 22287544
Authors: Gu, Z. - Liu, W. - Wei, J. - Yan, Z.
Journal: J Biol Chem

Emerging evidence suggests that metabotropic glutamate receptors (mGluRs) are potential novel targets for brain disorders associated with the dysfunction of prefrontal cortex (PFC), a region critical for cognitive and emotional processes. Since NMDAR dysregulation has been strongly associated with the pathophysiology of mental illnesses, we examined the possibility that mGluRs might be involved in modulating PFC functions by targeting postsynaptic NMDARs. We found that application of prototypical group III mGluR agonists significantly reduced NMDAR-mediated synaptic and ionic currents in PFC pyramidal neurons, which was mediated by mGluR7 localized at postsynaptic neurons and involved the beta-arrestin/ERK signaling pathway. The mGluR7 modulation of NMDAR currents was prevented by agents perturbing actin dynamics, and by the inhibitor of cofilin, a major actin depolymerizing factor. Consistently, biochemical and immunocytochemical results demonstrated that mGluR7 activation increased cofilin activity and F-actin depolymerization via an ERK-dependent mechanism. Furthermore, mGluR7 reduced the association of NMDARs with the scaffolding protein PSD-95 and the surface level of NMDARs in an actin-dependent manner. These data suggest that mGluR7, by affecting the cofilin/actin signaling, regulates NMDAR trafficking and function. Since ablation of mGluR7 leads to a variety of behavioral symptoms related to PFC dysfunction, such as impaired working memory and reduced anxiety and depression, our results provide a potential mechanism for understanding the role of mGluR7 in mental health and disorders.

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Characterization and purification of a Na+/Ca2+ exchanger from an Archaebacterium.

Publication Date: 2012 Jan 27 PMID: 22287543
Authors: Mercado Besserer, G. - Nicoll, D. A. - Abramson, J. - Philipson, K. D.
Journal: J Biol Chem

The superfamily of cation/Ca2+ exchangers includes both Na+/Ca2+ exchangers (NCX) and Na+/Ca2+,K+ exchangers (NCKX) as the families characterized in most detail. These Ca2+ transporters have prominent physiological roles. For example, NCX and NCKX are important in regulation of cardiac contractility and visual processes, respectively. The superfamily also has a large number of members of a YrbG family expressed in prokaryotes. However, no members of this family have been functionally expressed and their transport properties are unknown. We have expressed, purified, and characterized a member of the YrbG family, MaX1 from Methanosarcinia acetivorans. MaX1 catalyzes Ca2+ uptake into membrane vesicles. The Ca2+ uptake requires intravesicular Na+ and is stimulated by an inside positive membrane potential. Despite very limited sequence similarity, MaX1 is a Na+/Ca2+ exchanger with kinetic properties similar to NCX. The availability of a prokaryotic Na+/Ca2+ exchanger should facilitate structural and mechanistic investigations.

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NT-PGC-1alpha is sufficient to link beta3-adrenergic receptor activation to the transcriptional and physiological components of adaptive thermogenesis.

Publication Date: 2012 Jan 26 PMID: 22282499
Authors: Chang, J. S. - Fernand, V. - Zhang, Y. - Shin, J. - Jun, H. J. - Joshi, Y. - Gettys, T. W.
Journal: J Biol Chem

PGC-1alpha is an inducible transcriptional coactivator that regulates cellular energy metabolism and adaptation to environmental and nutritional stimuli. In tissues expressing PGC-1alpha, alternative splicing produces a truncated protein (NT-PGC-1alpha) corresponding to the first 267 amino acids of PGC-1alpha. Brown adipose tissue also expresses two novel exon1b-derived isoforms of PGC-1alpha and NT-PGC-1alpha, which are 4 and 13 amino acids shorter in the N-termini than canonical PGC-1alpha and NT-PGC-1alpha, respectively. To evaluate the ability of NT-PGC-1alpha to substitute for PGC-1alpha and assess the isoform-specific role of NT-PGC-1alpha, adaptive thermogenic responses of adipose tissue were evaluated in mice lacking full length PGC-1alpha (FL-PGC-1-/-) but expressing slightly shorter but functionally equivalent forms of NT-PGC-1alpha (NT-PGC-1alpha254). At room temperature, NT-PGC-1alpha and NT-PGC-1alpha254 were produced from conventional exon 1a-derived transcripts in BAT of wild type and FL-PGC-1alpha-/- mice, respectively. However, cold exposure shifted transcription to exon 1b, increasing exon 1b-derived mRNA levels. The resulting transcriptional responses produced comparable increases in energy expenditure and maintenance of core body temperature in WT and FL-PGC-1alpha-/- mice. Moreover, treatment of the two genotypes with a selective beta3-adrenergic receptor agonist produced similar increases in energy expenditure, mitochondrial DNA, and reductions in adiposity. Collectively, these findings illustrate that the transcriptional and physiological responses to sympathetic input are unabridged in FL-PGC-1alpha-/- mice, and that NT-PGC-1alpha is sufficient to link beta3-AR activation to adaptive thermogenesis in adipose tissue. Furthermore, the transcriptional shift from exon 1a to 1b isoform-specific roles for NT-PGC-1alpha in basal and adaptive thermogenesis.

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EphA signaling promotes actin-based dendritic spine remodeling through the slingshot phosphatase.

Publication Date: 2012 Jan 26 PMID: 22282498
Authors: Zhou, L. - Jones, E. V. - Murai, K. K.
Journal: J Biol Chem

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre-/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.

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Cytochrome b mutation Y268S conferring the atovaquone resistance phenotype in the malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression.

Publication Date: 2012 Jan 26 PMID: 22282497
Authors: Fisher, N. - Abd Majid, R. - Antoine, T. - Al-Helal, M. - Warman, A. J. - Johnson, D. J. - Lawrenson, A. S. - Ranson, H. - O'Neill, P. M. - Ward, S. A. - Biagini, G. A.
Journal: J Biol Chem

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g MalaroneTM) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc1 complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Qo pocket of cytochrome b, most notably near the conserved Pro260-Glu261-Trp262-Tyr263 (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterisation of an atovaquone resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270 fold shift the inhibitory constant (Ki) for atovaquone with a concomitant reduction in the Vmax of the bc1 complex of ~40% and a 3 fold increase in the observed Km for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein (ISP) content in Y268S bc1 suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared to the 3D7 (atovaquone sensitive) control strain in bc1 and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes off-sets the fitness cost resulting from reduced bc1 activity.

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Biochemical analysis of the human mismatch repair proteins hMutSalpha MSH2G674A-MSH6 and MSH2-MSH6T1219D.

Publication Date: 2012 Jan 25 PMID: 22277660
Authors: Geng, H. - Sakato, M. - Derocco, V. - Yamane, K. - Du, C. - Erie, D. A. - Hingorani, M. - Hsieh, P.
Journal: J Biol Chem

The heterodimeric human MSH2-MSH6 (hMutSalpha) protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2 G674A or Msh6 T1217D mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have reduced lifespan due to loss of the MMR pathway (1, 2). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSalpha proteins, MSH2G674A-MSH6wt and MSH2wt-MSH6T1219D, are profiled in a variety of functional assays and, as expected, fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2wt-MSH6T1219D fails to couple nucleotide binding and mismatch recognition; whereas, MSH2G674A-MSH6wt has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

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PARAMECIUM BURSARIA CHLORELLA VIRUS 1 ENCODES A POLYAMINE ACETYLTRANSFERASE.

Publication Date: 2012 Jan 25 PMID: 22277659
Authors: Charlop-Powers, Z. - Jakoncic, J. - Gurnon, J. R. - Van Etten, J. L. - Zhou, M. M.
Journal: J Biol Chem

Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study we report a 1.5A crystal structure of PBCV1 A654L in a complex with coenzyme A. The structure reveals A654L's unique feature that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named vPAT, acetylates polyamines such as putrescine, spermidine, cadaverine and homosperimidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication.

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Crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction.

Publication Date: 2012 Jan 25 PMID: 22277658
Authors: Fan, C. - Chou, C. Y. - Tong, L. - Xiang, S.
Journal: J Biol Chem

Urea carboxylase (UC) is conserved in many bacteria, algae and fungi, and catalyzes the conversion of urea to allophanate, an essential step in the utilization of urea as a nitrogen source in these organisms. UC belongs to the biotin-dependent carboxylase superfamily, and shares the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains with these other enzymes, but its carboxyltransferase (CT) domain is distinct. Currently there is no information on the molecular basis of catalysis by UC. We report here crystal structure of the Kluyveromyces lactis UC and biochemical studies to assess the structural information. Structural and sequence analyses indicate the CT domain of UC belongs to a large family of proteins with diverse functions, including the Bacillus subtilis KipA-KipI complex, which has important functions in sporulation regulation. A structure of the KipA-KipI complex is not currently available, and our structure provides a framework to understand the function of this complex. Most interestingly, in the structure the CT domain interacts with the BCCP domain, with biotin and a urea molecule bound at its active site. This structural information and our follow-up biochemical experiments provided molecular insights into the UC carboxyltransfer reaction. Several structural elements important for the UC carboxyltransfer reaction are found in other biotin-dependent carboxylases and might be conserved within this family, and our data could shed light on the mechanism of these enzymes' catalysis.

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Cytosolic Bax: Does it require binding proteins to keep its pro-apoptotic activity in check?

Publication Date: 2012 Jan 25 PMID: 22277657
Authors: Vogel, S. - Raulf, N. - Bregenhorn, S. - Biniossek, M. L. - Maurer, U. - Czabotar, P. - Borner, C.
Journal: J Biol Chem

Bax is kept inactive in the cytosol by refolding its C-terminal transmembrane domain into the hydrophobic binding pocket. Although energetic calculations predicted this conformation to be stable, numerous Bax binding proteins were reported and suggested to further stabilize inactive Bax. Unfortunately, most of them have not been validated in a physiological context on the endogenous level. Here we use gel filtration analysis of the cytosol of primary and established cells to show that endogenous, inactive Bax runs 20-30 kD higher than recombinant Bax, suggesting Bax dimerization or the binding of a small protein. Dimerization was excluded by a lack of interaction of differentially tagged Bax proteins, and by comparing the sizes of dimerized recombinant Bax with cytosolic Bax on blue native gels. Surprisingly, when analyzing cytosolic Bax complexes by high sensitivity mass spectrometry after anti-Bax immuno-precipitation or consecutive purification by gel filtration and blue native gel electrophoresis, we detected only one protein, called p23 hsp90 co-chaperone, which consistently and specifically co-purified with Bax. However, this protein could not be validated as a crucial inhibitory Bax binding partner as its over- or underexpression did not show any apoptosis defects. By contrast cytosolic Bax exhibits a slight molecular mass shift on SDS-PAGE as compared to recombinant Bax, which suggests a posttranslational modification and/or a structural difference between the two proteins. We propose that in most healthy cells, cytosolic endogenous Bax is a monomeric protein that does not necessarily need a binding partner to keep its pro-apoptotic activity in check.

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Sphingoid bases and the serine catabolic enzyme CHA1 define a novel Feedforward/Feedback mechanism in the response to serine availability.

Publication Date: 2012 Jan 25 PMID: 22277656
Authors: Montefusco, D. J. - Newcomb, B. - Gandy, J. L. - Brice, S. E. - Matmati, N. - Cowart, L. A. - Hannun, Y. A.
Journal: J Biol Chem

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases PHS and DHS as the likely mediators of Cha1 upregulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 upregulation via the transcription factor Cha4. Since the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Delta strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Delta strain displayed a significant growth defect that was sphingolipid dependent. Together this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate upregulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

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Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C-terminus.

Publication Date: 2012 Jan 25 PMID: 22277655
Authors: von Loehneysen, K. - Noack, D. - Hayes, P. - Friedman, J. S. - Knaus, U. G.
Journal: J Biol Chem

Redox regulation of signaling molecules contributes critically to propagation of intracellular signals. The main source providing reactive oxygen species (ROS) for these physiological processes are activated NADPH oxidases (Nox/Duox family). In a pathophysiological context, some NADPH oxidase complexes produce large amounts of ROS either as part of the antimicrobial immune defense or as pathologic oxidative stress in many chronic diseases. Thus, understanding the switch from a dormant, inactive conformation to the active state of these enzymes will aid the development of inhibitors. As exogenously expressed Nox4 represents the only constitutively active enzyme in this family, analysis of structural determinants that permit this active conformation was undertaken. Our focus was directed towards a cell-based analysis of the first intracellular loop, the B-loop, and the C-terminus, two regions of Nox family enzymes that are essential for electron transfer. Mutagenesis of the B-loop identified several unique residues and a polybasic motif that contribute to the catalytic activity of Nox4. By using a multifaceted approach including Nox4-Nox2 chimeras, mutagenesis and insertion of Nox2 domains, we show here that the penultimate 22 amino acids of Nox4 are involved in constitutive ROS generation. The appropriate spacing of the C-terminal Nox4 sequence may cooperate with a discrete arginine-based interaction site in the B-loop, providing an intrinsically active interface that could not be disrupted by peptides derived from the Nox4 C-terminus. These results indicate that accessibility for a Nox4-specific peptide inhibitor might be difficult to achieve in vivo.

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Identification of small molecule inhibitors of neurite loss induced by Abeta peptide using high-content screening.

Publication Date: 2012 Jan 25 PMID: 22277654
Authors: Ofengeim, D. - Shi, P. - Miao, B. - Fan, J. - Xia, X. - Fan, Y. - Lipinski, M. M. - Hashimoto, T. - Polydoro, M. - Yuan, J. - Wong, S. T. - Degterev, A.
Journal: J Biol Chem

Multiple lines of evidence indicate a strong relationship between Abeta peptide-induced neurite degeneration and the progressive loss of cognitive functions in Alzheimer's disease (AD) patients and in AD animal models. This prompted us to develop a high content screening assay (HCS) and Neurite Image Quantitator (NeuriteIQ) software to quantify the loss of neuronal projections induced by Abeta peptide neurons and enable us to identify new classes of neurite-protective small molecules, which may represent new leads for AD drug discovery. We identified thirty-six inhibitors of Abeta-induced neurite loss in the 1,040 compound National Institute of Neurological Disorders and Stroke (NINDS) custom collection of known bioactives and FDA approved drugs. Activity clustering showed that non-steroidal anti-inflammatory drugs (NSAIDs) were significantly enriched among the hits. Notably, NSAIDs have previously attracted significant attention as potential drugs for AD, however their mechanism of action remains controversial. Our data revealed that cyclooxygenase-2 (COX-2) expression was increased following Abeta treatment. Furthermore, multiple distinct classes of COX inhibitors efficiently blocked neurite loss in primary neurons, suggesting that increased COX activity contributes to Abeta peptide-induced neurite loss. Finally, we discovered that the detrimental effect of COX activity on neurite integrity may be mediated through the inhibition of peroxisome proliferator-activated receptor gamma (PPARgamma activity. Overall, our work establishes the feasibility of identifying small molecule inhibitors of Abeta induced neurite loss using the NeuriteIQ pipeline and provides novel insights into the mechanisms of neuroprotection by NSAIDs.

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Distinct peptide-binding specificities of SH3 domains can be determined by modulation of local energetics across the binding interface.

Publication Date: 2012 Jan 25 PMID: 22277653
Authors: Gorelik, M. - Davidson, A. R.
Journal: J Biol Chem

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behaviour, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it to the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. While the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

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Solid-support EPR studies of Abeta40 monomers reveal a structured state with three ordered segments.

Publication Date: 2012 Jan 25 PMID: 22277652
Authors: Gu, L. - Ngo, S. - Guo, Z.
Journal: J Biol Chem

Alzheimer's disease is associated with the pathological accumulation of amyloid-beta peptide (Abeta) in the brain. Soluble Abeta oligomers formed during early aggregation process are believed to be neurotoxins and causative agents in Alzheimer's. Abeta monomer is the building block for amyloid assemblies. A comprehensive understanding of the structural features of Abeta monomer is crucial for delineating the mechanism of Abeta oligomerization. Here we investigated the structures of Abeta40 monomer using a solid-support approach, in which Abeta40 monomers are tethered on the solid support via an N-terminal His-tag to prevent further aggregation. EPR spectra of tethered Abeta40 with spin labels at 18 different positions show that Abeta40 monomers adopt a completely disordered structure under denaturing conditions. Under native conditions, however, EPR spectra suggest that Abeta40 monomers adopt both a disordered state and a structured state. The structured state of Abeta40 monomer has three more ordered segments at 14-18, 29-30, and 38-40. Interactions between these segments may stabilize the structured state, which likely plays an important role in Abeta aggregation.

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Ligand-dependent corepressor (LCoR) recruitment by Kruppel-like factor 6 (KLF6) regulates expression of the cyclin-dependent kinase inhibitor CDKN1A Gene.

Publication Date: 2012 Jan 25 PMID: 22277651
Authors: Calderon, M. R. - Verway, M. - An, B. S. - Difeo, A. - Bismar, T. A. - Ann, D. K. - Martignetti, J. A. - Shalom-Barak, T. - White, J. H.
Journal: J Biol Chem

The widely expressed transcriptional coregulator, ligand-dependent corepressor (LCoR), initially characterized as a regulator of nuclear receptor-mediated transactivation, functions through recruitment of C-terminal Binding Proteins (CtBPs) and histone deacetylases (HDACs) to its N-terminal and central domains, respectively. We performed a yeast two-hybrid screen for novel cofactors, and identified an interaction between the C-terminal domain of LCoR and the transcription factor Kruppel-like Factor 6 (KLF6), a putative tumor suppressor in prostate cancer. Subsequent experiments have revealed LCoR regulation of several KLF6 target genes notably p21WAF1/CIP1 (CDKN1A) and to a lesser extent E-cadherin (CDH1), indicating that LCoR regulates gene transcription through multiple classes of transcription factors. In multiple cancer cells, LCoR and KLF6 bind together on the promoters of the genes encoding CDKN1A and CDH1. LCoR contributes to KLF6-mediated transcriptional repression in a promoter- and cell type-dependent manner. Its inhibition of reporter constructs driven by the CDKN1A and CDH1 promoters in PC-3 prostate carcinoma cells is sensitive to treatment with the HDAC inhibitor trichostatin A (TSA). Additionally, the LCoR cofactor CtBP1 bound the same promoters and augmented the LCoR-dependent repression in PC-3 cells. Consistent with their inferred roles in transcriptional repression, siRNA-mediated knockdown of KLF6, LCoR or CtBP1 in PC-3 cells induced expression of CDKN1A and CDH1 and additional KLF6 target genes. We propose a novel model of LCoR function in which promoter-bound KLF6 inhibits transcription of the CDKN1A gene and other genes as well by tethering a transcriptional corepressor complex containing LCoR, with specific contributions by CtBP1 and HDACs.

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Normalization of cholesterol homeostasis by 2-hydroxypropyl-beta-cyclodextrin in neurons and glia from Niemann-Pick C1-deficient mice.

Publication Date: 2012 Jan 25 PMID: 22277650
Authors: Peake, K. B. - Vance, J. E.
Journal: J Biol Chem

Niemann-Pick C (NPC) disease is an inherited, progressive neurodegenerative disorder caused by mutations in the NPC1 or NPC2 gene that result in an accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/L) and impaired export of cholesterol from LE/L to the endoplasmic reticulum (ER). Recent studies demonstrate that administration of cyclodextrin (CD) to Npc1-/- mice eliminates cholesterol sequestration in LE/L of many tissues including the brain, delays neurodegeneration, and increases life-span of the mice. We have now investigated cholesterol homeostasis in NPC1-deficient cells of the brain in response to CD. Primary cultures of neurons and glial cells from Npc1-/- mice were incubated for 24 h with 0.1 to 10 mM CD after which survival and cholesterol homeostasis were monitored. Although 10 mM CD was profoundly neurotoxic, and altered astrocyte morphology, 0.1 and 1 mM CD were not toxic but effectively mobilized stored cholesterol from the LE/L as indicated by filipin staining. However, 0.1 and 1 mM CD altered cholesterol homeostasis in opposite directions. The data suggest that 0.1 mM CD releases cholesterol trapped in LE/L of neurons and astrocytes and increases cholesterol availability at the ER, whereas 1 mM CD primarily extracts cholesterol from the plasma membrane and reduces ER cholesterol. These studies in Npc1-/- neurons and astrocytes establish a dose of CD (0.1 mM) that would likely be beneficial in NPC disease. The findings are timely because treatment of NPC disease patients with CD is currently being initiated.

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Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis.

Publication Date: 2012 Jan 25 PMID: 22277649
Authors: Kobayashi, K. - Sudiarta, I. P. - Kodama, T. - Fukushima, T. - Ara, K. - Ozaki, K. - Sekiguchi, J.
Journal: J Biol Chem

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS-MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m)=4.8 mM and k(cat)=0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m)=12.3 mM and k(cat)=0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.

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Glutathione degradation by the alternative pathway (DUG pahway) in Saccharomyces cerevisiae is initiated by the (Dug2p-Dug3p)2 complex, a novel GATase enzyme acting on glutathione.

Publication Date: 2012 Jan 25 PMID: 22277648
Authors: Kaur, H. - Ganguli, D. - Bachhawat, A. K.
Journal: J Biol Chem

The recently identified, fungal-specific alternative pathway of glutathione degradation requires the participation of three genes DUG1, DUG2 and DUG3. Dug1p has earlier been shown to function as a Cys-Gly specific dipeptidase. In the present study we describe the characterization of Dug2p and Dug3p. Dug3p has a functional glutamine amidotransferase (GATase) II domain that is catalytically important for glutathione degradation as demonstrated through mutational analysis. Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. Dug2p was also shown to be able to homodimerize and this was mediated by its M20A peptidase domain. In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. The functional complex had a molecular weight that corresponded to (Dug2p-Dug3p)(2) in addition to higher molecular weight oligomers, and displayed Michaelis-Menten kinetics. (Dug2p-Dug3p)(2) had a Km for glutathione of 1.2 mM, suggesting a novel GATase enzyme that acted on glutathione. Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. The DUG2 and DUG3 genes, but not DUG1, were derepressed by sulphur limitation. Based on these studies and the functioning of GATases, a mechanism is proposed for the functioning of the Dug proteins in the degradation of glutathione.

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Impaired transforming growth factor-beta (TGF-beta) transcriptional activity and cell proliferation control of a menin in-frame deletion mutant associated with multiple endocrine neoplasia type 1 (MEN1).

Publication Date: 2012 Jan 24 PMID: 22275377
Authors: Canaff, L. - Vanbellinghen, J. F. - Kaji, H. - Goltzman, D. - Hendy, G. N.
Journal: J Biol Chem

Multiple Endocrine Neoplasia type 1 (MEN1) is characterized by tumors of the parathyroid, enteropancreas and anterior pituitary. The MEN1 gene encodes the tumor suppressor menin of 610 amino acids that has multiple protein partners and activities. The particular pathways, that when lost, lead to tumorigenesis. are not known. We demonstrated that members of a three-generation MEN1 kindred are heterozygous for a donor splice-site mutation at the beginning of intron 3 (IVS3+1G>A). Lymphoblastoid cells of a mutant gene carrier had, in addition to the wild-type menin transcript, an aberrant transcript resulting from use of a cryptic splice-site within exon III that splices to the start of exon IV. The predicted menin Delta(184-218) mutant has an in-frame deletion of 35 amino acids, but is otherwise of wild-type sequence. The transfected menin Delta(184-218) mutant was well expressed and fully able to mediate the normal inhibition of the activity of the transcriptional regulators JunD and NF-kappaB. However, it was defective in mediating TGF-beta-stimulated Smad3 action in promoter-reporter assays in insulinoma cells. Importantly, lymphoblastoid cells from an individual heterozygous for the mutation had reduced TGF-beta-induced (Smad3) transcriptional activity, but normal JunD and NF-kappaB function. In addition, the mutant gene carrier lymphoblastoid cells proliferated faster and were less responsive to the cytostatic effects of TGF-beta than cells from an unaffected family member. In conclusion, the menin mutant exhibits selective loss of the TGF-beta signaling pathway and loss of cell proliferation control contributing to the development of MEN1.

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A novel RhoA/ROCK- CPI-17 -MEF2C signaling pathway regulates vascular smooth muscle cell gene expression.

Publication Date: 2012 Jan 23 PMID: 22275376
Authors: Pagiatakis, C. - Gordon, J. W. - Ehyai, S. - McDermott, J. C.
Journal: J Biol Chem

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1alpha (PP1alpha), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1alpha inhibition resulted in increased expression of myocardin, while ectopic expression of PP1alpha inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of the PP1alpha inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1alpha, and CPI-17.

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Structural basis for Ca2+-induced activation and dimerization of estrogen receptor alpha by calmodulin.

Publication Date: 2012 Jan 23 PMID: 22275375
Authors: Zhang, Y. - Li, Z. - Sacks, D. B. - Ames, J. B.
Journal: J Biol Chem

The estrogen receptor alpha (ER-a) regulates expression of target genes implicated in development, metabolism, and breast cancer. Calcium-dependent regulation of ER-a is critical for activating gene expression and is controlled by calmodulin (CaM). Here we present the NMR structures for the two lobes of CaM each bound to a functional fragment of ER-a (residues 287-305). A model of the complete CaM/ER-a complex was constructed by combining these 2 structures with additional data. The two lobes of CaM both compete for binding at the same site on ER-a (residues 292, 296, 299, 302 and 303), which explains why full-length CaM binds two molecules of ER-a in a 1:2 complex and stabilizes ER-a dimerization. Exposed glutamate residues in CaM (E11, E14, E84, E87) form salt bridges with key lysine residues in ER-a (K299, K302 and K303), which is likely to prevent ubiquitination at these sites and inhibit degradation of ER-a. Mutants of ER-a at the CaM binding site (W292A and K299A) weaken binding to CaM, and I298E/K299D disrupts estrogen-induced transcription. CaM facilitates dimerization of ER-a in the absence of estrogen, and stimulation of ER-a by either Ca2+ and/or estrogen may serve to regulate transcription in a combinatorial fashion.

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The mechanism of fragility at BCL2 minor breakpoint cluster region during t(14;18) chromosomal translocation.

Publication Date: 2012 Jan 24 PMID: 22275374
Authors: Nambiar, M. - Raghavan, S. C.
Journal: J Biol Chem

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at 3 prime UTR of BCL2 gene or downstream and are mainly clustered in either major breakpoint region or minor breakpoint cluster region (mcr). The RAG complex induces breaks at IgH locus of chromosome 14, while mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time we show that RAGs can nick mcr, however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg2+ and Mn2+ are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at mcr are independent of nonamer, while a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggests a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

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Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

Publication Date: 2012 Jan 24 PMID: 22275373
Authors: Chen, S. - Gulla, S. - Cai, C. - Balk, S. P.
Journal: J Biol Chem

Our previous findings indicated that androgen receptor (AR) phosphorylation at Serine 81 (S81) is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that S81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G1 or S phase did not disrupt androgen-induced S81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at S81 and activated by the transcriptional CDK9. To assess the function of S81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand-switch strategy using a ligand binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR regulated PSA or TMPRSS2 genes. Chromatin immunoprecipitation (ChIP) showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for S81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR S81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that S81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription, and suggest that CDK1 mediated S81 phosphorylation during mitosis provides a pool of S81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

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Nrf2 up-regulates anti-apoptotic protein Bcl-2 and prevents cellular apoptosis.

Publication Date: 2012 Jan 25 PMID: 22275372
Authors: Niture, S. K. - Jaiswal, A. K.
Journal: J Biol Chem

Nuclear transcription factor Nrf2 regulates expression and coordinated induction of a battery of genes encoding cytoprotective and drug-transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival and increased drug resistance. In this report, we investigated the role of Nrf2 in up-regulation of anti-apoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells exposed to antioxidant tert-butylhydroquinone (t-BHQ) led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element (ARE) between nucleotides -3148 to -3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/super shift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 ARE. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2 resulting in increased Bcl-2 level, decreased etoposide and UV/gamma radiation mediated DNA fragmentation. In addition, siRNA-mediated down regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knock down of Bcl-2 in Nrf2 activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and groth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance.

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The growth-suppressive function of the Polycomb group protein Polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain.

Publication Date: 2012 Jan 24 PMID: 22275371
Authors: Robinson, A. K. - Leal, B. Z. - Chadwell, L. V. - Wang, R. - Ilangovan, U. - Kaur, Y. - Junco, S. E. - Schirf, V. - Osmulski, P. A. - Gaczynska, M. - Hinck, A. P. - Demeler, B. - McEwen, D. G. - Kim, C. A.
Journal: J Biol Chem

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared to when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared to no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer deficient Ph mutants. This data provides the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Since the SAM linker had not been previously considered important for the function of SAM containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins in order to regulate a diverse array of intracellular functions.

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Antibody-induced enhancement of factor VIIa activity through distinct allosteric pathways.

Publication Date: 2012 Jan 24 PMID: 22275370
Authors: Andersen, L. M. - Andreasen, P. A. - Svendsen, I. - Keemink, J. - Ostergaard, H. - Persson, E.
Journal: J Biol Chem

In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation. The antibodies could be classified into two groups based on their patterns of affinities for different conformations of FVIIa. Whereas one class of antibodies affected both the Km and kcat the other class mainly affected the Km. The antibody-induced activity enhancement could be traced to maturation of the substrate binding pockets and the oxyanion hole, evident by an increased affinity for p-aminobenzamidine, an increased rate of antithrombin inhibition, an increased rate of incorporation of diisopropylfluorophosphate, and an enhanced fraction of molecules with a buried N-terminus of the catalytic domain in the presence of antibodies. As demonstrated by site-directed mutagenesis, the two groups of antibodies appear to have overlapping, although clearly different, epitopes in the 170-loop. Our findings suggest that binding of ligands to specific residues in the 170-loop or its spatial vicinity may stabilize the S1 pocket and the oxyanion hole and they may have general implications for the molecular understanding of FVIIa regulatory mechanisms.

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Insulin promotes neuronal survival via the alternatively spliced protein kinase C delta II (PKCdeltaII) isoform.

Publication Date: 2012 Jan 24 PMID: 22275369
Authors: Apostolatos, A. - Song, S. - Acosta, S. - Peart, M. - Watson, J. E. - Bickford, P. - Cooper, D. R. - Patel, N. A.
Journal: J Biol Chem

Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. Protein kinase C delta (PKCdelta) plays a central role in cell survival. Its splice variants, PKCdeltaI and PKCdeltaII, are switches that determine cell survival and fate. PKCdeltaI promotes apoptosis while PKCdeltaII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCdeltaII isoform in HT22 cells. The expression of PKCdeltaI splice variant remains unchanged. Insulin increases PKCdeltaII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCdeltaII alternative splicing. Using over-expression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and Bcl-xL via PKCdeltaII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells over-expressing PKCdeltaII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCdeltaII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.

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Stable incorporation of ATPase subunits into 19S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails.

Publication Date: 2012 Jan 24 PMID: 22275368
Authors: Lee, S. H. - Moon, J. H. - Yoon, S. K. - Yoon, J. B.
Journal: J Biol Chem

The 26S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19S RP, implying that the 20S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.

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Affixing the N-terminal alpha helix of the voltage dependent anion channel to the channel's wall does not prevent its voltage gating.

Publication Date: 2012 Jan 24 PMID: 22275367
Authors: Teijido, O. - Ujwal, R. - Hillerdal, C. O. - Kullman, L. - Rostovtseva, T. K. - Abramson, J.
Journal: J Biol Chem

The Voltage Dependent Anion Channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The 3D structure of VDAC1 reveals a channel formed by 19 beta-strands and an N-terminal alpha-helix located near the midpoint of the pore. The position of this alpha-helix causes a narrowing of the cavity but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established but the molecular mechanism remains debated; yet, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal alpha-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 (mVDAC1) that cross links the alpha-helix to the wall of the beta-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified mVDAC1 exhibited typical voltage gating. These results suggest that the N-terminal alpha-helix is located inside the pore of VDAC in the open state and remains associated with beta-strand 11 of the pore wall during voltage gating.

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Connections between Sphingosine Kinase and Phospholipase D in the Abscisic Acid Signaling Pathway in Arabidopsis.

Publication Date: 2012 Jan 24 PMID: 22275366
Authors: Guo, L. - Mishra, G. - Markham, J. E. - Li, M. - Tawfall, A. - Welti, R. - Wang, X.
Journal: J Biol Chem

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) both are lipid messengers involved in plant response to abscisic acid (ABA). Our previous data indicate that PA binds to sphingosine kinase (SPHK) and increases its phyto-S1P-producing activity. To understand the cellular and physiological functions of the PA-SPHK interaction, we isolated Arabidopsis thaliana SPHK mutants sphk1-1 and sphk2-1 and characterized them, together with phospholipase Dalpha1 knockout, pldalpha1, in plant response to ABA. Compared to wild-type (WT) plants, the SPHK mutants and pldalpha1 all displayed decreased sensitivity to ABA-promoted stomatal closure. Phyto-S1P promoted stomatal closure in sphk1-1 and sphk2-1, but not in pldalpha1, whereas PA promoted stomatal closure in sphk1-1, sphk2-1, and pldalpha1. The ABA activation of PLDalpha1 in leaves and protoplasts was attenuated in the SPHK mutants, and the ABA activation of SPHK was reduced in pldalpha1. In response to ABA, the accumulation of long-chain base phosphate (LCBP) was decreased in pldalpha1 whereas PA production was decreased in SPHK mutants, compared WT. Collectively, these results indicate that SPHK and PLDalpha1 act together in ABA response and that SPHK and phyto-S1P act upstream of PLDalpha1 and PA in mediating the ABA response. PA is involved in the activation of SPHK, and activation of PLDalpha1 requires SPHK activity. The data suggest that SPHK/phyto-S1P and PLDa1/PA are co-dependent in amplification of response to ABA, mediating stomatal closure in Arabidopsis.

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Effect of carcinogenic acrolein on DNA repair and mutagenic susceptibility.

Publication Date: 2012 Jan 24 PMID: 22275365
Authors: Wang, H. T. - Hu, Y. - Tong, D. - Huang, J. - Gu, L. - Wu, X. R. - Chung, F. L. - Li, G. M. - Tang, M. S.
Journal: J Biol Chem

Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic alpha- and gamma-hydroxy-1, N2-cyclic propano-2-deoxyguanosine adducts (alpha-OH-Acr-dG and gamma-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair (BER) and mismatch repair (MMR). While Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2 and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor, MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA, but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.

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The RAD51 ATP cap regulates nucleoprotein filament stability.

Publication Date: 2012 Jan 24 PMID: 22275364
Authors: Amunugama, R. - He, Y. - Willcox, S. - Forties, R. A. - Shim, K. S. - Bundschuh, R. - Luo, Y. - Griffith, J. - Fishel, R.
Journal: J Biol Chem

RAD51 mediates homologous recombination (HR) by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP gamma-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(D316) with lysine [HsRAD51(D316K)] decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) [MvRAD51(D302K)] and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(D316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity.

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Lipocalin-type prostaglandin D2 synthase regulates glial cell migration and morphology through marcks: prostaglandin D2-independent effects.

Publication Date: 2012 Jan 24 PMID: 22275363
Authors: Lee, S. - Jang, E. - Kim, J. H. - Kim, J. H. - Lee, W. H. - Suk, K.
Journal: J Biol Chem

Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH2 to PGD2. Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD2-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. The expression of l-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of Akt, RhoA, and Jnk pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD2 products. L-PGDS protein interacted with myristoylated alanine-rich C-kinase substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.

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C1q/TNF-related protein-12 (CTRP12), a novel adipokine that improves insulin sensitivity and glycemic control in mouse models of obesity and diabetes.

Publication Date: 2012 Jan 24 PMID: 22275362
Authors: Wei, Z. - Peterson, J. M. - Lei, X. - Cebotaru, L. - Wolfgang, M. J. - Baldeviano, G. C. - Wong, G. W.
Journal: J Biol Chem

Despite the prevalence of insulin resistance and Type 2 diabetes mellitus (T2DM), their underlying mechanisms remain incompletely understood. Many secreted endocrine factors and the inter-tissue crosstalk they mediate are known to be dysregulated in T2DM. Here, we describe CTRP12, a novel adipokine with anti-diabetic actions. The mRNA and circulating levels of CTRP12 were decreased in a mouse model of obesity, but its expression in adipocytes was increased by the anti-diabetic drug rosiglitazone. A modest rise in circulating levels of CTRP12 by recombinant protein administration was sufficient to lower blood glucose in wild-type, leptin-deficient ob/ob, and diet-induced obese (DIO) mice. A short-term elevation of serum CTRP12 by adenovirus-mediated expression improved glucose tolerance and insulin sensitivity, normalized hyperglycemia and hyperinsulinemia, and lowered postprandial insulin resistance in obese and diabetic mice. CTRP12 improves insulin sensitivity in part by enhancing insulin signaling in the liver and adipose tissue. Further, CTRP12 also acts in an insulin-independent manner; in cultured hepatocytes and adipocytes, CTRP12 directly activated the PI3K-Akt signaling pathway to suppress gluconeogenesis and promote glucose uptake, respectively. Collectively, these data establish CTRP12 as a novel metabolic regulator linking adipose tissue to whole-body glucose homeostasis through insulin-dependent and independent mechanisms.

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Hepatocyte-specific deletion of Janus kinase 2 (JAK2) protects against diet-induced steatohepatitis and glucose intolerance.

Publication Date: 2012 Jan 24 PMID: 22275361
Authors: Shi, S. Y. - Garcia Martin, R. - Duncan, R. E. - Choi, D. - Lu, S. Y. - Schroer, S. A. - Cai, E. P. - Luk, C. T. - Hopperton, K. E. - Domenichiello, A. F. - Tang, C. - Naples, M. - Dekker, M. J. - Giacca, A. - Adeli, K. - Wagner, K. U. - Bazinet, R. P. - Woo, M.
Journal: J Biol Chem

Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and is now considered to be the hepatic manifestation of the metabolic syndrome. However, the role of steatosis per se and the precise factors required in the progression to steatohepatitis or insulin resistance remain elusive. The JAK-STAT pathway is critical in mediating signaling of a wide variety of cytokines and growth factors. Mice with hepatocyte-specific deletion of Janus kinase 2 (L-JAK2 KO mice) develop spontaneous steatosis as early as two weeks of age. In this study, we investigated the metabolic consequences of jak2 deletion in response to diet-induced metabolic stress. To our surprise, despite the profound hepatosteatosis, deletion of hepatic jak2 did not sensitize the liver to accelerated inflammatory injury on a prolonged high fat diet (HFD). This was accompanied by complete protection against HFD-induced whole-body insulin resistance and glucose intolerance. Improved glucose-stimulated insulin secretion and an increase in beta-cell mass were also present in these mice. Moreover, L-JAK2 KO mice had progressively reduced adiposity in association with blunted hepatic growth hormone signaling. These mice also exhibited increased resting energy expenditure on both chow and high fat diet. In conclusion, our findings indicate a key role of hepatic JAK2 in metabolism such that its absence completely arrests steatohepatitis development and confers protection against diet-induced systemic insulin resistance and glucose intolerance.

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Picomolar concentrations of free zinc(II) ions regulate receptor protein tyrosine phosphatase beta activity.

Publication Date: 2012 Jan 24 PMID: 22275360
Authors: Wilson, M. - Hogstrand, C. - Maret, W.
Journal: J Biol Chem

As key enzymes in the regulation of biological phosphorylations, protein tyrosine phosphatases are central to the control of cellular signaling and metabolism. Zinc(II) ions are known to inhibit these enzymes but the physiological significance of this inhibition has remained elusive. Employing metal buffering for strict metal control and performing a kinetic analysis, we now demonstrate that zinc(II) ions are mixed-type reversible inhibitors of the cytoplasmic catalytic domain of the receptor protein tyrosine phosphatase beta (RPTPbeta, VEPTP). The Ki(Zn) value is 28 +/- 4 pM, six orders of magnitude lower than zinc inhibition reported previously for this enzyme. It exceeds the affinity of the most potent synthetic small molecule inhibitors targeting these enzymes. Inhibition is in the range of cellular zinc(II) ion concentrations, suggesting that zinc regulates this enzyme, which is involved in vascular physiology and angiogenesis. Thus, for some enzymes that are not recognized as zinc metalloenzymes, zinc binding inhibits rather than activates as in classical zinc enzymes. Activation then requires removal of the inhibitory zinc.

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Insulin-like growth factor receptor-1 and nuclear factor kappa B are crucial survival signals that regulate caspase-3 mediated lens epithelial cell differentiation initiation.

Publication Date: 2012 Jan 24 PMID: 22275359
Authors: Basu, S. - Rajakaruna, S. - Menko, A. S.
Journal: J Biol Chem

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation-induction was dependent on an Insulin-like Growth Factor Receptor-1 (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling expression of molecules in the Bcl-2 and Inhibitor of Apoptosis Protein (IAP) families. This effect of IGF-1R was mediated through Nuclear Factor Kappa B (NFkappaB), demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFkappaB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFkappaB signaling pathway redirected cell fate from differentiation towards apoptosis. These results provided the first evidence that the IGF-1R/NFkappaB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.

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The role of receptor-attached phosphates in the binding of visual and non-visual arrestins to G protein-coupled receptors.

Publication Date: 2012 Jan 24 PMID: 22275358
Authors: Diaz Gimenez, L. E. - Kook, S. - Vishnivetskiy, S. A. - Ahmed, M. R. - Gurevich, E. V. - Gurevich, V. V.
Journal: J Biol Chem

Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated ac-tive receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initi-ating G protein-independent signaling. The inter-action of rhodopsin-attached phosphates with Lys14 and Lys15 in the beta-strand I was shown to disrupt the interaction of alpha-helix I, beta-strand I, and the C-tail of visual arrestin-1 , facilitating its tran-sition into active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants where both were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phos-phorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by bioluminescence resonance energy transfer (BRET) based assay. BRET data confirmed the role of Lys14 and Lys15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins, where the K2A mu-tations had little effect on net BRETMAX values for the M2 muscarinic acetylcholine (M2R), beta2-adrenergic (beta2AR), or D2-dopamine (D2R) recep-tors. Moreover, phosphorylation deficient mutant of M2R interacted with wild type non-visual ar-restins normally, whereas phosphorylation-deficient beta2AR mutants bound arrestins at 20-50% level of wild type beta2AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. While arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of ar-restin-2 and -3 is much greater in case of beta2AR than M2R and D2R.

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Mislocalization of fukutin protein by disease-causing missense mutations can be rescued with treatments directed at folding amelioration.

Publication Date: 2012 Jan 24 PMID: 22275357
Authors: Tachikawa, M. - Kanagawa, M. - Yu, C. C. - Kobayashi, K. - Toda, T.
Journal: J Biol Chem

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of alpha-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. While the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of alpha-dystroglycan have been reported, both in Japan and elsewhere. To better understand the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and alpha-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R and Y371C instead accumulated in the ER. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low-temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of alpha-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.

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Critical role of O-GlcNAc transferase in prostate cancer invasion, angiogenesis and metastasis.

Publication Date: 2012 Jan 24 PMID: 22275356
Authors: Lynch, T. P. - Ferrer, C. M. - Jackson, S. R. - Shahriari, K. S. - Vosseller, K. - Reginato, M. J.
Journal: J Biol Chem

Cancer cells universally increase glucose and glutamine consumption leading to the altered metabolic state known as the Warburg effect; one metabolic pathway, highly dependent on glucose and glutamine, is the Hexosamine Biosynthetic Pathway (HBP). Increased flux through the HBP leads to increases in the post-translation addition of O-linked-beta-N-acetylglucosamine (O-GlcNAc) on various nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer and, recently, O-GlcNAcylation was shown to play a role in breast cancer; however, O-GlcNAcylation in other cancers remains poorly defined. Here, we show O-GlcNAc transferase (OGT) is overexpressed in prostate cancer compared to normal prostate epithelium, and OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines. Reducing O-GlcNAcylation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and vascular endothelial growth factor (VEGF), resulting in inhibition of invasion and angiogenesis. OGT-mediated regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, a key regulator of invasion and angiogenesis, as reducing OGT expression led to increased FoxM1 protein degradation. Conversely, overexpression of a FoxM1 degradation-resistant mutant abrogated OGT RNAi-mediated effects on invasion, MMP levels, angiogenesis, and VEGF expression. Using a mouse model of metastasis, we found reduction of OGT expression blocked bone metastasis. Altogether, these data suggest that, as prostate cancer cells alter glucose and glutamine levels, O-GlcNAc modifications and OGT levels become elevated and are required for regulation of malignant properties, implicating OGT as a novel therapeutic target in the treatment of cancer.

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Mycobacterial induction of autophagy varies by species and occurs independently of mTOR inhibition.

Publication Date: 2012 Jan 24 PMID: 22275355
Authors: Zullo, A. J. - Lee, S.
Journal: J Biol Chem

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to latent infection. While many factors are involved, the mammalian autophagy pathway is recognized as a determinant that can influence the course of infection. Intervention aimed at utilizing autophagy to clear infection requires an examination of the autophagy and signal transduction induced by mycobacteria under native conditions. With both pathogenic and non-pathogenic mycobacteria, we show that infection correlates with an increase in the mammalian target of rapamycin (mTOR) activity indicating that autophagy induction by mycobacteria occurs in an mTOR independent manner. Analysis of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guerin (BCG), which respectively induces high and low autophagy responses, indicates that lipid material is capable of inducing both autophagy and mTOR signaling. Although mycobacterial infection potently induces mTOR activity, we confirm that bacterial viability can be reduced by rapamycin treatment. In addition, our work demonstrates that BCG can reduce autophagy responses to M. smegmatis suggesting that specific mechanisms are used by BCG to minimize host cell autophagy. We conclude that autophagy induction and mTOR signaling take place concurrently during mycobacterial infection and that host autophagy responses to any given mycobacterium stem from multiple factors, including the presence of activating macromolecules and inhibitory mechanisms.

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Critical role of an N-terminal end-localized nuclear export signal in regulation of activating transcription factor 2 (ATF2) subcellular localization and transcriptional activity.

Publication Date: 2012 Jan 24 PMID: 22275354
Authors: Hsu, C. C. - Hu, C. D.
Journal: J Biol Chem

Activating transcription factor 2 (ATF2) belongs to the basic leucine zipper (bZIP) family of transcription factors. ATF2 regulates target gene expression by binding to the cyclic AMP response element (CRE) as a homodimer or a heterodimer with c-Jun. Cytoplasmic localization of ATF2 was observed in melanoma, brain tissue from patients with Alzheimers disease, prostate cancer specimens and ionizing radiation-treated prostate cancer cells, suggesting that alteration of ATF2 subcellular localization may be involved in the pathogenesis of these diseases. We previously demonstrated that ATF2 is a nucleocytoplasmic shuttling protein and it contains two nuclear localization signals (NLS) in the basic region and one nuclear export signal (NES) in the leucine zipper domain (named LZ-NES). In the present study, we demonstrate that a hydrophobic stretch in the N terminus, (1)MKFKLHV(7), also functions as an NES (termed N-NES) in a chromosome region maintenance 1 (CRM1)-dependent manner. Mutation of both N-NES and LZ-NES results in a predominant nuclear localization whereas mutation of each individual NES only partially increases the nuclear localization. These results suggest that cytoplasmic localization of ATF2 requires function of at least one of the NESs. Further, mutation of N-NES enhances the transcriptional activity of ATF2, suggesting that the novel NES negatively regulates the transcription potential of ATF2. Thus, ATF2 subcellular localization is likely modulated by multiple mechanisms, and further understanding of the regulation of ATF2 subcellular localization under various pathological conditions will provide insight into the pathophysiological role of ATF2 in human diseases.

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Novel roles for the E3 ubiquitin ligase atrophin-interacting protein 4 and signal transduction adaptor molecule 1 in G protein-coupled receptor signaling.

Publication Date: 2012 Jan 24 PMID: 22275353
Authors: Malik, R. - Soh, U. J. - Trejo, J. - Marchese, A.
Journal: J Biol Chem

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease, however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Over-expression of an AIP4 catalytically inactive mutant and a mutant which shows poor binding to STAM-1 fail to enhance CXCR4-induced ERK-1/2 signaling, as compared to wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.

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Identification and structure determination of a novel anti-inflammatory mediator resolvin E3: 17,18-Dihydroxy-eicosapentaenoic acid.

Publication Date: 2012 Jan 24 PMID: 22275352
Authors: Isobe, Y. - Arita, M. - Matsueda, S. - Iwamoto, R. - Fujihara, T. - Nakanishi, H. - Taguchi, R. - Masuda, K. - Sasaki, K. - Urabe, D. - Inoue, M. - Arai, H.
Journal: J Biol Chem

Bioactive mediators derived from omega-3 eicosapentaenoic acid (EPA) elicit potent anti-inflammatory actions. Here we identified novel EPA metabolites including 8,18-dihydroxy-eicosapentaenoic acid (8,18-diHEPE), 11,18-diHEPE, 12,18-diHEPE, and 17,18-diHEPE from 18-HEPE. Unlike resolvins E1 and E2, both of which are biosynthesized by neutrophils via 5-lipoxygenase pathway, these metabolites are biosynthesized by eosinophils via 12/15-lipoxygenase pathway. Among them, two stereoisomers of 17,18-diHEPE, collectively termed resolvin E3 (RvE3), displayed a potent anti-inflammatory action by limiting neutrophil infiltration in zymosan-induced peritonitis. The planar structure of RvE3 was unambiguously determined to be 17,18-dihydroxy-5Z,8Z,11Z,13E,15E-EPE by high-resolution NMR, and the two stereoisomers were assigned to have 17,18R- and 17,18S-dihydroxy groups, respectively, using chemically synthesized 18R- and 18S-HEPE as precursors. Both 18R- and 18S-RvE3 inhibited neutrophil chemotaxis in vitro at low nanomolar concentrations. These findings suggest that RvE3 contributes to the beneficial actions of EPA in controlling inflammation and related diseases.

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High-throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

Publication Date: 2012 Jan 23 PMID: 22275351
Authors: Fueller, F. - Jehle, B. - Putzker, K. - Lewis, J. D. - Krauth-Siegel, R. L.
Journal: J Biol Chem

In African trypanosomes, the detoxification of broad-spectrum hydroperoxides relies on a unique cascade composed of trypanothione [T(SH)2], trypanothione reductase (TR), tryparedoxin (Tpx) and nonselenium glutathione peroxidase-type enzymes (Px). All three proteins are essential for Trypanosoma brucei. Here we subjected the complete system to a high-throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. Except one, all of them carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited TR but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx modifying Cys40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as target in the intact parasites. T(SH)2, present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC50-values down to <1 microM and exerted up to 83-fold lower toxicity towards HeLa cells. Irreversible inhibitors are traditionally regarded as unfavourable. However, a large number of antimicrobials and anticancer therapeutics act covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)2/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors, renders the parasite-specific oxidoreductase an attractive drug target molecule.

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The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP.

Publication Date: 2012 Jan 23 PMID: 22270372
Authors: Niwa, H. - Ewens, C. A. - Tsang, C. - Yeung, H. O. - Zhang, X. - Freemont, P. S.
Journal: J Biol Chem

p97/VCP is a type II AAA ATPase that forms a homohexamer with each protomer containing an N-terminal domain, two ATPase domains, D1 and D2, and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease Inclusion Body Myopathy associated with Paget Disease of Bone and Frontotemporal Dementia (IBMPFD), the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N- domain with D1. Here we have tested ten major IBMPFD-linked mutants for ATPase activity and found that all have increased activity over wild type, with one mutant, p97A232E, having three times higher activity. Further mutagenesis of p97A232E shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a co-planar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97A232E suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N- domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.

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Enhancers located in the heavy chain regulatory region (hs3a, hs1,2, hs3b, hs4) are dispensable for the diversity of VDJ recombination.

Publication Date: 2012 Jan 23 PMID: 22270371
Authors: Rouaud, P. - Vincent-Fabert, C. - Fiancette, R. - Cogne, M. - Pinaud, E. - Denizot, Y.
Journal: J Biol Chem

V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple immunoglobulin heavy chain (IgH) cis-regulatory elements control B-cell ontogeny. The intergenic control region 1 (IGCR1), the DQ52 promoter/enhancer and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination while there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B cell differentiation. By the contrary, the earliest antigen-independent steps of B-cell ontogeny would be only under control by the upstream Cmu elements of the locus.

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Heat shock protein 90 alpha (Hsp90alpha) is phosphorylated in response to DNA damage and accumulates in repair foci.

Publication Date: 2012 Jan 23 PMID: 22270370
Authors: Quanz, M. - Herbette, A. - Sayarath, M. - de Koning, L. - Dubois, T. - Sun, J. S. - Dutreix, M.
Journal: J Biol Chem

DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr7) residue of heat shock protein 90alpha (Hsp90alpha) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90alpha then accumulated at sites of DNA double-strand breaks (DSBs) and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90alpha was dependent on phosphatidylinositol-3 kinase-like kinases (PIKK), including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DSBs by non homologous end-joining (NHEJ) and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90alpha. In vivo, reverse-phase protein array data for tumors revealed that basal levels of Thr7-phosphorylated Hsp90alpha were correlated with phosphorylated histone H2AX levels. The Thr7 phosphorylation of the ubiquitously produced and secreted Hsp90alpha may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.

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Absence of post-phosphoryl modification in dystroglycanopathy mouse models and wild-type tissues expressing a non-laminin binding form of alpha-dystroglycan.

Publication Date: 2012 Jan 23 PMID: 22270369
Authors: Kuga, A. - Kanagawa, M. - Sudo, A. - Chan, Y. M. - Tajiri, M. - Manya, H. - Kikkawa, Y. - Nomizu, M. - Kobayashi, K. - Endo, T. - Lu, Q. L. - Wada, Y. - Toda, T.
Journal: J Biol Chem

Alpha-dystroglycan (alpha-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of alpha-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-alpha2,3-Gal-beta1,4-GlcNAc-beta1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of alpha-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, fukutin-related protein (FKRP) is also involved in the post-phosphoryl modification of O-mannose on alpha-DG. Furthermore, we have found that the glycosylation status of alpha-DG in lung and testis is minimally affected by defects in fukutin, LARGE or FKRP. alpha-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of alpha-DG functional expression as a laminin receptor in normal tissues and cells.

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Up-regulation of pro-apoptotic Bim and down-regulation of anti-apoptotic Mcl-1 cooperatively mediate the enhanced tumor cell death induced by the combination of a MEK inhibitor and a microtubule inhibitor.

Publication Date: 2012 Jan 23 PMID: 22270368
Authors: Kawabata, T. - Tanimura, S. - Asai, K. - Kawasaki, R. - Matsumaru, Y. - Kohno, M.
Journal: J Biol Chem

Blockade of the extracellular signal regulated kinase (ERK) signaling pathway by ERK kinase (MEK) inhibitors selectively enhances the induction of apoptosis by microtubule inhibitors in tumor cells in which this pathway is constitutively activated. We have now examined the mechanism by which such drug combinations induce enhanced cell death by applying time-lapse microscopy to track the fate of individual cells. MEK inhibitors did not affect the first mitosis after drug exposure, but most cells remained arrested in interphase without entering a second mitosis. Low concentrations of microtubule inhibitors induced prolonged mitotic arrest followed by exit of cells from mitosis without division, with most cells remaining viable. The combination of a MEK inhibitor and a microtubule inhibitor, however, induced massive cell death during prolonged mitosis. Impairment of spindle assembly checkpoint function by RNA interference (RNAi)-mediated depletion of Mad2 or BubR1 markedly suppressed such prolonged mitotic arrest and cell death. The cell death was accompanied by up-regulation of the pro-apoptotic protein Bim (to which MEK inhibitors contributed) and down-regulation of the anti-apoptotic protein Mcl-1 (to which microtubule and MEK inhibitors contributed synergistically). Whereas RNAi-mediated knockdown of Bim suppressed cell death, stabilization of Mcl-1 by RNAi-mediated depletion of Mule slowed its onset. Depletion of Mcl-1 sensitized tumor cells to MEK inhibitor-induced cell death, an effect that was antagonized by knockdown of Bim. The combination of MEK and microtubule inhibitors thus targets Bim and Mcl-1 in a cooperative manner to induce massive cell death in tumor cells with aberrant ERK pathway activation.

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Factor inhibiting HIF (FIH) recognises distinct molecular features within hypoxia inducible factor (HIF)-alpha versus ankyrin repeat substrates.

Publication Date: 2012 Jan 23 PMID: 22270367
Authors: Wilkins, S. E. - Karttunen, S. - Hampton-Smith, R. J. - Murchland, I. - Chapman-Smith, A. - Peet, D. J.
Journal: J Biol Chem

Factor Inhibiting HIF (FIH) catalyses the beta-hydroxylation of asparaginyl residues in hypoxia inducible factors (HIF)-alpha transcription factors, as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Whilst FIH-mediated hydroxylation of HIF-alpha is well characterised, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH-substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterise a conserved RLL motif in HIF, and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo, and therefore have important consequences for HIF regulation.

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ETS-1 regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell SKOV-3.

Publication Date: 2012 Jan 23 PMID: 22270366
Authors: Ghosh, S. - Basu, M. - Roy, S. S.
Journal: J Biol Chem

Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of MMPs are regulated by different cytokines and growth factors. VEGF, the potent angiogenic cytokine induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38MAPK as well as PI3K/AKT pathways. VEGF induces the expression of MMP-2, MMP-9 and MMP-13 and hence regulates the metastasis of SKOV-3 ovarian cancer cells and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38MAPK pathways. Interestingly, VEGF induces expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 binds to both MMP-9 and MMP-13 promoters. Further, VEGF acts through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knocking down of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriches ETS-1 in nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and nuclear localization was blocked by the specific inhibitors of the PI3K and p38MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38MAPK by VEGF results in ETS-1 gene expression, that activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.

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The contradictory functions (activation/termination) of neutrophil proteinase 3 (PR3) in IL-33 biological activity.

Publication Date: 2012 Jan 23 PMID: 22270365
Authors: Bae, S. - Kang, T. - Hong, J. - Lee, S. - Choi, J. - Jhun, H. - Kwak, A. - Hong, K. - Kim, E. - Jo, S. - Kim, S.
Journal: J Biol Chem

IL-1 family ligand does not possess a typical hydrophobic signal peptide and needs a processing enzyme for maturation. The maturation process of IL-33 (IL-1F11), a new member of the IL-1 family ligand, remains unclear. Precursor IL-33 ligand affinity column isolates neutrophil proteinase 3 (PR3) from human urinary proteins. PR3 is a known IL-1 family ligand processing enzyme for IL-1beta (IL-1F2) and IL-18 (IL-1F4) including other inflammatory cytokines. We investigated PR3 in the maturation process of precursor IL-33 since we isolated urinary PR3 by using precursor IL-33 ligand affinity column. PR3 converted inactive human and mouse precursor IL-33 proteins to biological active forms; however, the increase of PR3 incubation time abrogated IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, PR3 cut precursor IL-33 and IL-18 at various sites and yielded multi-bands. The increased incubation period of PR3 abated mature IL-33 in a time-dependent manner. The result is consistent with the decreased bioactivity of IL-33 along with the increased PR3 incubation time. Six different human and mouse rIL-33 proteins were expressed by the predicted consensus amino acid sequence of PR3 cleavage sites and tested for bioactivities. The human IL-33/p1 was highly active but human IL-33/p2 and p3 proteins were inactive. Our results suggest the dual functions (activation/termination) of PR3 in IL-33 biological activity.

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Integrin-mediated Membrane Blebbing is Dependent on Sodium-Proton Exchanger 1 and Sodium-Calcium Exchanger 1 Activity.

Publication Date: 2012 Jan 23 PMID: 22270364
Authors: Yi, Y. H. - Chang, Y. S. - Lin, C. H. - Lew, T. S. - Tang, C. Y. - Tseng, W. L. - Tseng, C. P. - Lo, S. J.
Journal: J Biol Chem

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.

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Characterization of the ArabinoGalactan protein 31 (AGP31) of Arabidopsis thaliana: new advances on the Hyp-O-glycosylation of the Pro-rich domain.

Publication Date: 2012 Jan 23 PMID: 22270363
Authors: Hijazi, M. - Durand, J. - Pichereaux, C. - Pont, F. - Jamet, E. - Albenne, C.
Journal: J Biol Chem

Proteins are important actors in plant cell walls since they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoproteins (HRGPs) constitute a complex family of O-glycoproteins with various structures and functions. In this study, we characterized an atypical HRGP, the ArabinoGalactan Protein 31 (AGP31) which displays a multi-domain organization unique in Arabidopsis thaliana, consisting of a short AGP motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the beta-glucosyl Yariv reagent, but interacting with the PeaNut Agglutinin (PNA) lectin was proposed. Besides, the N-terminal short AGP motif was assumed to be substituted by arabinogalactans (AGs). Altogether, AGP31 was found to be highly heterogeneous in cell walls since AGs could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion.

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Functional association of the catalytic and ancillary modules dictates enzymatic activity in a glycoside hydrolase family-43 beta-xylosidase.

Publication Date: 2012 Jan 23 PMID: 22270362
Authors: Morais, S. - Salama-Alber, O. - Barak, Y. - Hadar, Y. - Wilson, D. B. - Lamed, R. - Shoham, Y. - Bayer, E. A.
Journal: J Biol Chem

beta-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization and kinetic analysis of a glycoside hydrolase family-43 beta-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60C and 5.5 to 6, respectively, were determined. The apparent Km value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (kcat) was 6.72 s-1. T. fusca Xyl43A contains a catalytic module at the N-terminus and an ancillary module (termed herein Module-A) of undefined function at the C-terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Non-denaturing gel electrophoresis revealed a specific non-covalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity.

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Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion.

Publication Date: 2012 Jan 23 PMID: 22270361
Authors: Hanania, R. - Sun, H. S. - Xu, K. - Pustylnik, S. - Jeganathan, S. - Harrison, R. E.
Journal: J Biol Chem

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-gamma causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages upregulate MMP-9 expression and secretion upon immunological challenge, and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution, as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and PDI, in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs, and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.

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Sometimes it takes two to tango: contributions of dimerization to functions of human alpha-defensin HNP1.

Publication Date: 2012 Jan 23 PMID: 22270360
Authors: Pazgier, M. - Wei, G. - Ericksen, B. - Jung, G. - Wu, Z. - de Leeuw, E. - Yuan, W. - Szmacinski, H. - Lu, W. Y. - Lubkowski, J. - Lehrer, R. I. - Lu, W.
Journal: J Biol Chem

Human myeloid alpha-defensins called HNPs play multiple roles in innate host defense. The Trp26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild-type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by X-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile20 dramatically reduced the ability of HNP1 to kill S. aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill E. coli. The W26A and MeIle20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp26-mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile20 and Leu25. The present studies demonstrate that dimerization contributes to some, but not all of the many and varied activities of HNP1.

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Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires beta-catenin activation via Akt/GSK3-dependent and MTA1/Wnt1-dependent pathways.

Publication Date: 2012 Jan 23 PMID: 22270359
Authors: Yan, D. - Avtanski, D. - Saxena, N. K. - Sharma, D.
Journal: J Biol Chem

Perturbations in the adipocytokines-profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1) and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of beta-catenin leading to increased promoter recruitment. Silencing of beta-catenin or treatment with small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3beta (GSK3beta) via Akt activation resulting in a substantial decrease in the formation of GSK3beta-LKB1-Axin complex which leads to increased accumulation of beta-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3beta phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3beta phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of Wnt/beta-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3beta phosphorylation and beta-catenin activation. Furthermore, analysis of leptin-treated breast tumors show increased expression of Wnt1, pGSK3beta and Vimentin along with higher nuclear accumulation of beta-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized crosstalk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.

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Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells.

Publication Date: 2012 Jan 23 PMID: 22270358
Authors: Hamada, H. - Kurusu, T. - Okuma, E. - Nokajima, H. - Kiyoduka, M. - Koyano, T. - Sugiyama, Y. - Okada, K. - Koga, J. - Saji, H. - Miyao, A. - Hirochika, H. - Yamane, H. - Murata, Y. - Kuchitsu, K.
Journal: J Biol Chem

Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knockout cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX -induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.

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Validating macromolecular structures.

Publication Date: 2012 Jan 20 PMID: 22267959
Authors: Allewell, N.
Journal: J Biol Chem

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Angiopoietin-like 4 (Angptl4) is a physiological mediator of intracellular lipolysis in murine Adipocytes.

Publication Date: 2012 Jan 19 PMID: 22267746
Authors: Gray, N. E. - Lam, L. N. - Yang, K. - Zhou, A. Y. - Koliwad, S. - Wang, J. C.
Journal: J Biol Chem

Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice,and TG hydrolysis induced by fasts of 6- or 24 hours was greatly reduced in mice lacking Angptl4 (Angptl4(-/-)). Glucocorticoid treatment mimicked the lipolytic effects of fasting, though with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this,cAMP levels and TG hydrolysis were reduced in primary (Angptl4(-/-)) murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4 (hANGPTL4). Remarkably, hANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4-modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance.

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Pannexin1 drives multicellular aggregate compaction via a signaling cascade that remodels the actin cytoskeleton.

Publication Date: 2012 Jan 20 PMID: 22267745
Authors: Bao, B. - Lai, C. P. - Naus, C. C. - Morgan, J. R.
Journal: J Biol Chem

Pannexin 1 (Panx1) is a novel gap junction protein shown to have tumor-suppressive properties. To model its in vivo role in the intra-tumor biomechanical environment, we investigated whether Panx1 channels modulate the dynamic assembly of multicellular C6 glioma aggregates. Treatment with carbenoxolone (CBX) and probenecid (PBN), which directly and specifically block Panx1 channels respectively, showed that Panx1 is involved in accelerating aggregate assembly. Experiments further showed that exogenous ATP can reverse the inhibitive effects of CBX and that aggregate compaction is sensitive to the purinergic antagonist suramin. With a close examination of the F-actin microfilament network, these findings show that Panx1 channels act as conduits for ATP secretion that stimulate the P(2)X(7) purinergic receptor pathway, in turn upregulating actomyosin function. Using a unique 3D scaffold-free method to quantify multicellular interactions, this study shows that Panx1 is intimately involved in regulating intercellular biomechanical interactions pivotal in the progression of cancer.

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Resolving Holliday Junctions with the Escherichia coli UvrD Helicase.

Publication Date: 2012 Jan 20 PMID: 22267744
Authors: Carter, A. S. - Tahmaseb, K. - Compton, S. A. - Matson, S. W.
Journal: J Biol Chem

The Escherichia coli UvrD helicase is known to function in the mismatch repair and nucleotide excision repair pathways and has also been suggested to have roles in recombination and replication restart. The primary intermediate DNA structure in these two processes is the Holliday junction. UvrD has been shown to unwind a variety of substrates including partial duplex DNA, nicked DNA, forked DNA structures, blunt duplex DNA and RNA-DNA hybrids. Here we demonstrate that UvrD also catalyzes the robust unwinding of Holliday junction substrates. To characterize this unwinding reaction we have employed steady state helicase assays, pre-steady state rapid quench helicase assays, DNaseI footprinting and electron microscopy. We conclude that UvrD binds initially to the junction, as compared to binding one of the blunt ends of the four-way junction to initiate unwinding, and resolves the synthetic substrate into two double-stranded fork structures. We suggest that UvrD, along with its mismatch repair partners, MutS and MutL, may utilize its ability to directly unwind Holliday junctions in the prevention of homeologous recombination. UvrD may also be involved in the resolution of stalled replication forks by unwinding the Holliday junction intermediate to allow bypass of the blockage.

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Identification of a nuclear localization sequence in beta-arrestin1 and its functional implications.

Publication Date: 2012 Jan 21 PMID: 22267743
Authors: Hoeppner, C. Z. - Cheng, N. - Ye, R. D.
Journal: J Biol Chem

A mounting body of evidence suggests that beta-arrestin1 plays important roles in the nucleus, but how beta-arrestin1 enters the nucleus remains unclear since no nuclear import signal has been identified in the beta-arrestins. We sought to characterize the cellular localization of wild type beta-arrestin1 and a series of N domain mutants to determine the structural basis and functional implications of beta-arrestin1 nuclear localization. A seven-residue candidate nuclear localization sequence (NLS) was identified based on sequence analysis. Mutation of the NLS led to a loss of beta-arrestin1 nuclear localization in transfected cells. Exogenous expression of wild type beta-arrestin1 enhanced the transcriptional activity of nuclear factor kappaB (NF-kB) induced by bradykinin, while mutation of the NLS reduced this effect by two thirds relative to wild type controls. Loss of beta-arrestin1 nuclear localization was accompanied by reduced recruitment of the CREB binding protein and altered post-translational modification profile of p65/RelA. Further mutational analysis identified Lys157 within the putative NLS as being critical to nuclear localization of beta-arrestin1. Substitution of Lys157 to Ala led to reduced nuclear localization, decreased promoter binding by p65/RelA and decreased IL-1beta gene transcription. These results demonstrate a critical role for beta-arrestin1 nuclear localization in scaffolding and transcriptional regulation.

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Controlling lipid fluxes at the glycerol 3-phosphate acyltransferase step in yeast: Unique contribution of Gat1p to oleic acid-induced lipid particle formation.

Publication Date: 2012 Jan 21 PMID: 22267742
Authors: Marr, N. - Foglia, J. - Terebiznik, M. - Athenstaedt, K. - Zaremberg, V.
Journal: J Biol Chem

The ability to channel excess fatty acids into neutral lipids like triacylglycerol (TAG) is a critical strategy used by cells to maintain lipid homeostasis. Upon activation to acyl-CoA fatty acids become readily available as substrates for acyltransferases involved in neutral lipid synthesis. Neutral lipids are then packed into organelles derived from the endoplasmic reticulum (ER) called lipid particles (LPs). The first acylation step in the de novo pathway for TAG synthesis is catalyzed by glycerol 3-phosphate acyltransferases (GPATs). Two isoforms, Gat1p/Gpt2p and Gat2p/Sct1p, are present in the yeast Saccharomyces cerevisiae. Previous evidence indicated that these enzymes contribute differentially to the synthesis of TAG in actively growing cells. In this work we studied the role of the yeast GPATs in the formation of LPs induced by a surplus of oleic acid. Yeast lacking Gat1p (but not Gat2p) were sensitive to oleate and failed to accumulate LPs induced by this unsaturated fatty acid. It is shown that oleate induces dephosphorylation of Gat1p as well as an increment in its levels. Most importantly, we identified novel Gat1p crescent structures that are formed in the presence of oleate. These structures are connected with the ER and are intimately associated with LPs. No such structures were observed for Gat2p. A crucial point of control of lipid fluxes at the GPAT step is proposed.

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Identification of a grass-specific enzyme that acylates monolignols with p-coumarate.

Publication Date: 2012 Jan 21 PMID: 22267741
Authors: Withers, S. - Lu, F. - Kim, H. - Zhu, Y. - Ralph, J. - Wilkerson, C. G.
Journal: J Biol Chem

Lignin is a major component of plant cell walls that is essential to its function. However, the strong bonds that bind the various subunits of lignin and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol and produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin and these monolignol conjugates can also be monomer-precursors of lignin. While monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters.

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Incompatibility between a pair of residues from the pre-M1 linker and Cys-loop blocks surface expression of the glycine receptor.

Publication Date: 2012 Jan 20 PMID: 22267740
Authors: Shan, Q. - Lynch, J. W.
Journal: J Biol Chem

Regulation of cell membrane excitability can be achieved either by modulating the functional properties of cell membrane-expressed single channels or by varying the number of expressed channels. Whereas the structural basis underlying single channel properties has been intensively studied, the structural basis contributing to surface expression is less well characterized. Here we demonstrate that homologous substitution of the pre-M1 linker from the beta subunit prevents surface expression of the alpha1 glycine receptor chloride channel. By investigating a series of chimeras comprising alpha1 and beta subunits, we hypothesized that this effect was due to incompatibility between a pair of positively-charged residues, which lie in close proximity to each other in the tertiary structure, from the pre-M1 linker and Cys-loop. Abolishing either positive charge restored surface expression. We propose that incompatibility (electrostatic repulsion) between this pair of residues misfolds the glycine receptor, and in consequence, the protein is retained in the cytoplasm and prevented from surface expression by the quality control machinery. This hypothesis suggests a novel mechanism, i.e. residue incompatibility, for explaining the mutation-induced reduction in channel surface expression, often present in the cases of hereditary hyperekplexia.

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Structural determinants of the Clostridium difficile toxin A glucosyltransferase activity.

Publication Date: 2012 Jan 20 PMID: 22267739
Authors: Pruitt, R. N. - Chumbler, N. M. - Rutherford, S. A. - Farrow, M. A. - Friedman, D. B. - Spiller, B. - Lacy, D. B.
Journal: J Biol Chem

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain (GTD) in the presence and absence of the co-substrate UDP-glucose. While the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable to TcdB in its modification of Rho-family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap-family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and GTD release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.

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Biochemical characterization of the ribosome assembly GTPase RbgA in Bacillus subtilis.

Publication Date: 2012 Jan 20 PMID: 22267738
Authors: Achila, D. - Gulati, M. - Jain, N. - Britton, R. A.
Journal: J Biol Chem

The ribosome biogenesis GTPase A protein (RbgA) is involved in the assembly of the large ribosomal subunit in Bacillus subtilis and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45S in sucrose density gradients. Here we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow kcat (14 h-1) and has a high Km (90 muM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K+ ions for GTPase activity, which was confirmed experimentally. Interaction with 50S subunits, but not 45S intermediates, increases GTPase activity ~55 fold. Stable association with 50S subunits and 45S intermediates was nucleotide dependent and GDP did not support strong interaction with either of the subunits. GTP and GMPPNP were sufficient to promote association with the 45S intermediate while only GMPPNP was able to support binding to the 50S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.

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Structure of Complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of the Membrane Attack Complex (MAC).

Publication Date: 2012 Jan 20 PMID: 22267737
Authors: Aleshin, A. E. - Schraufstatter, I. U. - Stec, B. - Bankston, L. A. - Liddington, R. C. - Discipio, R. G.
Journal: J Biol Chem

The complement Membrane Attack Complex (MAC) is formed by the sequential assembly of C5b with 4 homologous proteins: one copy each of C6, C7 and C8, and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MACPF domain flanked by 4-9 auxiliary domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8alphabetagamma heterodimer and perforin show that the central domain of C6 adopts a closed (perforin-like) state that is distinct from the open conformations in C8. We further show that C6, C8alpha and C8beta contain 3 homologous subdomains (upper, lower and regulatory) related by rotations about 2 hinge-points. In C6, the regulatory segment comprises 4 auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8beta, rotation of the regulatory segment is linked to an opening of the central beta-sheet of its clockwise partner, C8alpha. These observations lead us to propose a model for initiation and unidirectional propagation of MAC assembly in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating opening of the MACPF beta-sheet of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins (CDCs), but is distinct from that recently proposed for perforin.

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Inhibin alpha-Subunit N-terminus Interacts With Activin Type IB receptor To Disrupt Activin Signaling.

Publication Date: 2012 Jan 20 PMID: 22267736
Authors: Zhu, J. - Lin, S. J. - Zou, C. - Makanji, Y. - Jardetzky, T. S. - Woodruff, T. K.
Journal: J Biol Chem

Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin beta-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin alpha-subunit mature domain (N-terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the alpha-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the alpha-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free alpha-subunit inhibited activin signaling in a pituitary gonadotrope derived cell line (LbetaT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An alpha-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free alpha-subunit in LbetaT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the alpha-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.

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A novel role for galectin-8 as a mediator of coagulation factor V endocytosis by megakaryocytes.

Publication Date: 2012 Jan 20 PMID: 22267735
Authors: Zappelli, C. - van der Zwaan, C. - Thijssen-Timmer, D. C. - Mertens, K. - Meijer, A. B.
Journal: J Biol Chem

Galectin-8 (Gal8) interacts with beta-galactoside-containing glycoproteins, and has recently been implicated to play a role in platelet activation. It has been suggested that Gal8 may also interact with platelet coagulation factor V (FV). This indispensable cofactor is stored in alpha-granules of platelets via a poorly understood endocytic mechanism that only exists in megakaryocytes (platelet precursor cells). In this study, we now assessed the putative role of Gal8 for FV biology. Surface plasmon resonance analysis and a solid phase binding assay revealed that Gal8 binds FV. The data further show that beta-galactosides block the interaction between FV and Gal8. These findings indicate that Gal8 specifically interacts with FV in a carbohydrate-dependent manner. Confocal microscopy studies and flow cytometry analysis demonstrated that megakaryocytic DAMI cells internalize FV. Flow cytometry showed that these cells express Gal8 on their cell surface. Reducing the functional presence of Gal8 on the cells by either an anti-Gal8 antibody or by siRNA technology markedly impaired the endocytic uptake of FV. Compatible with the apparent role of Gal8 for FV uptake, endocytosis of FV was also affected in the presence of beta-galactosides. Strikingly, TPO differentiated DAMI cells, which represent a more mature megakaryocytic state, not only loose the capacity to express cell-surface bound Gal8 but also the ability to internalize FV. Collectively, our data reveal a novel role for the tandem-repeat Gal8 in promoting FV endocytosis.

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Biochemical inhibition of ATase1 and ATase2 activity reduces BACE1 levels and Abeta generation.

Publication Date: 2012 Jan 20 PMID: 22267734
Authors: Ding, Y. - Ko, M. H. - Pehar, M. - Kotch, F. - Peters, N. R. - Luo, Y. - Salamat, S. M. - Puglielli, L.
Journal: J Biol Chem

The cellular levels of beta-site APP cleaving enzyme 1 (BACE1), the rate-limiting enzyme for the generation of the Alzheimer's disease (AD) amyloid beta-peptide (Abeta), are tightly regulated by two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2. Here we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of first and second generation compounds that inhibit ATase1/ATase2 and down-regulate the expression levels as well as activity of BACE1. The mechanism of action involves competitive and non-competitive inhibition as well as generation of unstable intermediates of the ATases that undergo degradation.

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Involvement of the stress kinase mitogen-activated protein kinase kinase 7 in the regulation of the mammalian circadian clock.

Publication Date: 2012 Jan 20 PMID: 22267733
Authors: Uchida, Y. - Osaki, T. - Yamasaki, T. - Shimomura, T. - Hata, S. - Horikawa, K. - Shibata, S. - Todo, T. - Hirayama, J. - Nishina, H.
Journal: J Biol Chem

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes such as cell proliferation, apoptosis, differentiation and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of a MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1epsilon (CK1epsilon). Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.

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Tyrosines in the Carboxy-terminus of Syk once Phosphorylated Interact with Signaling Proteins including TULA-2, a Negative Regulator of Mast Cell Degranulation.

Publication Date: 2012 Jan 20 PMID: 22267732
Authors: de Castro, R. O. - Zhang, J. - Groves, J. R. - Barbu, E. A. - Siraganian, R. P.
Journal: J Biol Chem

Activation of the high affinity IgE receptor (FcepsilonRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the carboxy terminus of the protein tyrosine kinase Syk. Synthetic peptides representing the last ten amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2 and GADS and the proteins phosphatases SHIP-1 and UBASH3B (also known as TULA-2). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far westerns showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2 and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcepsilonRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-2gamma. There was parallel enhancement of the receptor-induced degranulation and NFAT or NFkappaB activation indicating that TULA-2 like SHIP-1 functions as a negative regulator of FcepsilonRI signaling in mast cells. Therefore the terminal tyrosines of Syk once phosphorylated bind complexes of proteins that are positive and negative regulators of signaling in mast cells.

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PRELP inhibits the formation of the complement membrane attack complex.

Publication Date: 2012 Jan 20 PMID: 22267731
Authors: Happonen, K. E. - Melin-Furst, C. - Saxne, T. - Heinegard, D. - Blom, A. M.
Journal: J Biol Chem

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA) the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b- binding protein, which was suggested to locally down regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3- convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.

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Inhibition of AMPKalpha by doxorubicin accentuates genotoxic stress and cell death in mouse embryonic fibroblasts: role of p53 and SIRT1.

Publication Date: 2012 Jan 20 PMID: 22267730
Authors: Wang, S. - Song, P. - Zou, M. H.
Journal: J Biol Chem

Doxorubicin, an anthracycline antibiotic, is widely used in cancer treatment. Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells. Although its side effects in non-carcinoma cells, especially in heart tissue, are well known, the molecular targets of doxorubicin are poorly characterized. Here, we report that doxorubicin inhibits AMP-activated protein kinase (AMPK) resulting in SIRT1 dysfunction and p53 accumulation. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) or H9C2 cardiomyocyte were exposed to doxorubicin at different doses and durations. Cell death and p53, SIRT1, and AMPK levels were examined by western blot. In MEFs, doxorubicin inhibited AMPK activation, increased cell death, and induced robust p53 accumulation. Genetic deletion of AMPKalpha1 reduced NAD+ levels and SIRT1 activity, and significantly increased the levels of p53 and cell death. Pre-activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside, or transfection with an adenovirus encoding a constitutively active AMPK (AMPK-CA) markedly reduced the effects of doxorubicin in MEFs from AMPKalpha1 knockout mice. Conversely, pre-inhibition of AMPK further sensitized MEFs to doxorubicin-induced cell death. Genetic knockdown of p53 protected both wild-type and AMPKalpha1-/- MEFs from doxorubicin-induced cell death. p53 accumulation in AMPKalpha1-/- MEFs was reversed by SIRT1 activation by resveratrol. Taken together, these data suggest that AMPK inhibition by doxorubicin causes p53 accumulation and SIRT1 dysfunction in MEFs, and further that pharmacological activation of AMPK might alleviate the side effects of doxorubicin.

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Curcumin prevents aggregation in alpha-synuclein by increasing the reconfiguration rate.

Publication Date: 2012 Jan 20 PMID: 22267729
Authors: Ahmad, B. - Lapidus, L. J.
Journal: J Biol Chem

alpha-synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation particularly at high temperatures. In this work we examine the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We find strong binding of curcumin to alpha-synuclein in the hydrophobic non-amyloid-beta component (NAC) region and a complete inhibition of oligomers or fibrils. We also find that the reconfiguration rate within the unfolded protein is significantly increased at high temperatures. We conclude that alpha-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same timescale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.

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Phosphorylation of the amyloid-beta peptide at serine 8 attenuates its clearance via insulin degrading and angiotensin converting enzymes.

Publication Date: 2012 Jan 20 PMID: 22267728
Authors: Kumar, S. - Singh, S. - Hinze, D. - Josten, M. - Sahl, H. G. - Siepmann, M. - Walter, J.
Journal: J Biol Chem

Accumulation of amyloid beta-peptides (Abeta) in the brain is a common pathological feature of Alzheimer's disease (AD). Aggregates of Abeta are neurotoxic and appear to be critically involved in the neurodegeneration during AD pathogenesis. Accumulation of Abeta could be caused by increased production as indicated by several mutations in the amyloid precursor protein or the gamma-secretase components presenilin-1 or -2 that cause familial early onset AD. However, recent data also indicate a decreased clearance rate of Abeta in AD brains. We recently demonstrated that Abeta undergoes phosphorylation by extracellular or cell surface localized protein kinase A, leading to increased aggregation. Here we provide evidence that phosphorylation of monomeric Abeta at serine residue 8 also decreases its clearance by microglial cells. By using mass spectrometry, we demonstrate that phosphorylation at serine 8 inhibits the proteolytic degradation of monomeric Abeta by the insulin degrading enzyme (IDE), a major Abeta degrading enzyme released from microglial cells. Phosphorylation also decreased the degradation of Abeta by the angiotensin converting enzyme (ACE). In contrast, Abeta degradation by plasmin was largely unaffected by phosphorylation. Thus, phosphorylation of Abeta could play a dual role in Abeta metabolism. It decreases its proteolytic clearance and also promotes its aggregation. The inhibition of extracellular Abeta phosphorylation, stimulation of cell-surface protease expression and/or increasing their activity could be explored to promote Abeta degradation in AD therapy or prevention.

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A Mitochondrial membrane complex that contains proteins necessary for tRNA import in Trypanosoma brucei.

Publication Date: 2012 Jan 20 PMID: 22267727
Authors: Seidman, D. - Johsnon, D. - Gerbasi, V. - Golden, D. - Orlando, R. - Hajduk, S.
Journal: J Biol Chem

The mitochondrial genome of Trypanosoma brucei does not contain genes encoding tRNAs, instead this protozoan parasite must import nuclear encoded tRNAs from the cytosol for mitochondrial translation. Previously, it has been shown that mitochondrial tRNA import requires ATP hydrolysis and a proteinaceous mitochondrial membrane component. However, little is known about the mitochondrial membrane proteins involved in tRNA binding and translocation into the mitochondrion. Here we report the purification of a mitochondrial membrane complex using tRNA affinity purification and have identified several protein components of the putative tRNA translocon by mass spectrometry. Using an in vivo tRNA import assay, in combination with RNA interference, we have verified that two of these proteins, Tb11.01.4590 and Tb09.v1.0420, are involved in mitochondrial tRNA import. Using PTP-tagged Tb11.01.4590, additional associated proteins were identified including Tim17 and other mitochondrial proteins necessary for mitochondrial protein import. Results presented here identify and validate two novel protein components of the putative tRNA translocon and provide additional evidence that mitochondrial tRNA and protein import have shared components in trypanosomes.

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Pyroglutamate A(beta) aggravates behavioral deficits in 5XFAD mice.

Publication Date: 2012 Jan 20 PMID: 22267726
Authors: Wittnam, J. L. - Portelius, E. - Zetterberg, H. - Gustavsson, M. K. - Schilling, S. - Koch, B. - Demuth, H. U. - Blennow, K. - Wirths, O. - Bayer, T. A.
Journal: J Biol Chem

Pyroglutamate-modified Abeta peptides at amino acid position three (AbetapE3-42) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). AbetapE3-42 is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated AbetapE3-42 levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces AbetapE3-42. TBA42 mice showed age-dependent behavioral deficits and AbetapE3-42- accumulation. The Abeta profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-truncated and modified Abeta peptides: Abeta1-42, Abeta1-40, AbetapE3-40, AbetapE3-42, Abeta3-42, Abeta4-42 and Abeta5-42. 5XFAD and TBA42 mice were then crossed to generate bigenic FAD42 mice. At six months of age, FAD42 mice showed an aggravated behavioral phenotype compared to single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that AbetapE3 levels were elevated in FAD42 mice. No change in Abetax-42 or other Abeta isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of AbetapE-42 in FAD42 mice.

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ERdj4 is a soluble endoplasmic reticulum DnaJ family protein that interacts with ERAD machinery.

Publication Date: 2012 Jan 20 PMID: 22267725
Authors: Lai, C. W. - Otero, J. H. - Hendershot, L. M. - Snapp, E.
Journal: J Biol Chem

Protein localization within cells regulates accessibility for interactions with cofactors and substrates. The endoplasmic reticulum (ER) BiP co-factor, ERdj4, is upregulated by ER stress and has been implicated in ER Associated Degradation (ERAD) of multiple unfolded secretory proteins. Several other ERdj family members tend to interact selectively with nascent proteins, presumably because those ERdj proteins associate with the Sec61 translocon that facilitates entry of nascent proteins into the ER. How ERdj4 selects and targets terminally misfolded proteins for destruction remains poorly understood. In this study, we determined properties of ERdj4 that might aid in this function. ERdj4 was reported to retain its signal sequence and to be resistant to mild detergent extraction, suggesting that it was an integral membrane protein. However, live cell photobleaching analyses of GFP-tagged ERdj4 revealed the protein exhibits diffusion coefficients uncommonly high for an ER integral membrane protein and more similar to the mobility of a soluble luminal protein. Biochemical characterization established the ERdj4 signal sequence is cleaved to yield a soluble protein. Importantly, we found that both endogenous and over-expressed ERdj4 associate with the integral membrane protein, Derlin-1. Our findings now directly link ERdj4 to the ERAD machinery and suggest a model in which ERjd4 could help recruit clients from throughout the ER to ERAD sites.

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Activation of sterol response element binding proteins (SREBP) in alveolar type II cells enhances lipogenesis causing pulmonary lipotoxicity.

Publication Date: 2012 Jan 20 PMID: 22267724
Authors: Plantier, L. - Besnard, V. - Xu, Y. - Ikegami, M. - Wert, S. E. - Hunt, A. N. - Postle, A. D. - Whitsett, J. A.
Journal: J Biol Chem

Pulmonary inflammation is associated with altered lipid synthesis and clearance related to diabetes, obesity, and various inherited metabolic disorders. In many tissues, lipogenesis is regulated at the transcriptional level by the activity of Sterol Response Element Binding Proteins (SREBP). The role of SREBP activation in the regulation of lipid metabolism in the lung was assessed in mice in which both Insig1 and Insig2, proteins that bind and inhibit SREBPs in the endoplasmic reticulum, were deleted in alveolar type 2 cells. While deletion of either Insig1 or Insig2 did not alter SREBP activity or lipid homeostasis, deletion of both Insig proteins (Insig1/2(/) mice) activated SREBP1, causing marked accumulation of lipids that consisted primarily of cholesterol esters and triglycerides in type 2 epithelial cells and alveolar macrophages. Neutral lipids accumulated in type 2 cells in association with the increase in mRNAs regulating fatty acid, cholesterol synthesis, and inflammation. While bronchoalveolar lavage fluid (BALF) phosphatidylcholine (PC) was modestly decreased, lung phospholipid content and lung function were maintained. Insig1/2(/) mice developed lung inflammation and airspace abnormalities associated with the accumulation of lipids in alveolar type 2 cells, alveolar macrophages and within alveolar spaces. Deletion of Insig1/2 activated SREBP enhancing lipogenesis in respiratory epithelial cells resulting in lipotoxicity related lung inflammation and tissue remodeling.

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Competitive binding of SecA and ribosomes to the SecYEG translocon.

Publication Date: 2012 Jan 20 PMID: 22267723
Authors: Wu, Z. C. - de Keyzer, J. - Kedrov, A. - Driessen, A. J.
Journal: J Biol Chem

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles, and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

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The proton glutamate E268 regulates transport probability of the anion-proton exchanger CIC-5.

Publication Date: 2012 Jan 20 PMID: 22267722
Authors: Grieschat, M. - Alekov, A. K.
Journal: J Biol Chem

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH leads to greater increase of currents upon exchanging extracellular Cl(-) for SCN(-). However, neither transport uncoupling, unitary current amplitudes nor the voltage dependence of the depolarization-induced activation were altered by low pHs. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H alter the ratio between ClC-5 transport and nonlinear capacitance indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents, together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate E211 by the mutation E211C abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P - a mutation that decreases the anion affinity of the central binding site - reduces also the internal pH dependence of ClC-5. These results support the notion that protonation of the gating glutamate E211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate E268.

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Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts.

Publication Date: 2012 Jan 18 PMID: 22262865
Authors: Lee, J. - Kim, D. - Choi, J. - Choi, H. - Ryu, J. - Jeong, J. - Park, E. J. - Kim, S. H. - Kim, S.
Journal: J Biol Chem

A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocytes differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using oil red O staining method. A single spot on thin-layer chromatography of chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha (C/EBPalpha), adipocyte fatty acid-binding protein 2 (aP2), sterol response element binding protein-1c (SREBP1c), and stearoyl-coenzyme A desaturase-1 (SCD-1), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA-binding activity of C/EBPbeta, and lowered the production level of CyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis.

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Structural and biochemical basis of lectin Yos9 dimerization and possible contribution to self-association of the HMG-CoA reductase degradation (HRD) ubiquitin-ligase complex.

Publication Date: 2012 Jan 18 PMID: 22262864
Authors: Hanna, J. - Schutz, A. - Zimmermann, F. - Behlke, J. - Sommer, T. - Heinemann, U.
Journal: J Biol Chem

In yeast, the membrane-bound HRD ubiquitin ligase complex is a key player of the ER-associated protein degradation pathway that targets misfolded proteins for proteolysis. Yos9, a component of the luminal sub-module of the ligase, scans proteins for specific oligo-saccharide modifications, which constitute a critical determinant of the degradation signal. Here, we report the crystal structure of the Yos9 domain that was previously suggested to confer binding to Hrd3, another component of the HRD complex. We observe an alphabeta-roll domain architecture and a dimeric assembly which is confirmed by analytical ultracentrifugation of both the crystallized domain and full-length Yos9. Our binding studies indicate that, instead of this domain, the N-terminal part of Yos9 including the mannose-6-phosphate receptor homology domain mediates the association with Hrd3 in vitro. Our results support the model of a dimeric state of the HRD complex and provide first-time evidence of self-association on its luminal side.

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alpha-enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps.

Publication Date: 2012 Jan 18 PMID: 22262863
Authors: Mori, Y. - Yamaguchi, M. - Terao, Y. - Hamada, S. - Ooshima, T. - Kawabata, S.
Journal: J Biol Chem

Streptococcus pneumoniae is the most common causative agent of community acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae alpha-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae alpha-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that alpha-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared to untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that alpha-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that alpha-enolase increased neutrophil extracellular traps dependent killing of S. pneumoniae in human blood. Moreover, pull-down assay and mass spectrometry results identified myoblast antigen 24.1D5 as an alpha-enolase-binding protein on human neutrophils, while flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that alpha-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to alpha-enolase of S. pneumoniae.

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Externalized glycolytic enzymes are novel, conserved, and early biomarkers of apoptosis.

Publication Date: 2012 Jan 18 PMID: 22262862
Authors: Ucker, D. S. - Jain, M. R. - Pattabiraman, G. - Palasiewicz, K. - Birge, R. B. - Li, H.
Journal: J Biol Chem

The intriguing cell biology of apoptotic cell death results in the externalization of numerous autoantigens on the apoptotic cell surface, including protein determinants for specific recognition, linked to immune responses. Apoptotic cells are recognized by phagocytes and trigger an active immunosuppressive response ( "innate apoptotic immunity" [IAI]) even in the absence of engulfment. IAI is responsible for the lack of inflammation associated normally with the clearance of apoptotic cells; its failure also has been linked to inflammatory and autoimmune pathology, including systemic lupus erythematosis (SLE) and rheumatic diseases. Apoptotic recognition determinants underlying IAI have yet to be identified definitively; we argue that these molecules are surface-exposed (during apoptotic cell death), ubiquitously-expressed, protease-sensitive, evolutionarily-conserved, and resident normally in viable cells ("SUPER"). Taking independent and unbiased quantitative proteomic approaches to characterize apoptotic cell surface proteins and identify candidate SUPER determinants, we made the surprising discovery that components of the glycolysis pathway are enriched on the apoptotic cell surface. Our data demonstrate that glycolytic enzyme externalization is a common and early aspect of cell death in different cell types triggered to die with distinct suicidal stimuli. Exposed glycolytic enzyme molecules meet the criteria for IAI-associated SUPER determinants. In addition, our characterization of the apoptosis-specific externalization of glycolytic enzyme molecules may provide insight into the significance of previously reported cases of plasminogen binding to alpha-enolase on mammalian cells, as well as mechanisms by which commensal bacteria and pathogens maintain immune privilege.

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Structure-activity analysis of the dermcidin-derived peptide DCD 1L, an anionic antimicrobial peptide present in human sweat.

Publication Date: 2012 Jan 18 PMID: 22262861
Authors: Paulmann, M. - Arnold, T. - Linke, D. - Ozdirekcan, S. - Kopp, A. - Gutsmann, T. - Kalbacher, H. - Wanke, I. - Schuenemann, V. J. - Habeck, M. - Burck, J. - Ulrich, A. S. - Schittek, B.
Journal: J Biol Chem

Dermcidin encodes the anionic amphiphilic peptide DCD 1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD 1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis which is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD 1L forms oligomeric complexes which are stabilized by Zn2+. DCD-1L is able to form ion channels in the bacterial membrane and we propose that Zn2+-induced self-assembly of DCD 1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.

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Functional characterization of a novel aquaporin from Dictyostelium discoideum amoebae implies a unique gating mechanism.

Publication Date: 2012 Jan 18 PMID: 22262860
Authors: von Buelow, J. - Mueller-Lucks, A. - Kai, L. - Bernhard, F. - Beitz, E.
Journal: J Biol Chem

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP ), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wildtype AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N-terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude, that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

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The C-terminal proteolytic fragment of the breast cancer susceptibility type I protein (BRCA1) is degraded by the N-end rule pathway.

Publication Date: 2012 Jan 18 PMID: 22262859
Authors: Xu, Z. - Payoe, R. - Fahlman, R. P.
Journal: J Biol Chem

The breast cancer susceptibility type 1 gene product (BRCA1) is cleaved by caspases upon the activation of apoptotic pathways. After proteolysis the C-terminal fragment has been reported to translocate to the cytoplasm and promote cell death. Here we report that the C-terminal fragment is unstable in cells as it is targeted for degradation by the N-End rule pathway. The data reveals that mutating the wildtype N-terminal aspartate, of the C-terminal fragment, to valine stabilizes the fragment. If the N-terminus is mutated to another N-terminal destabilizing residue, like arginine, the C-terminal fragment remains unstable in cells. Lastly, the C-terminal fragment of BRCA1 is stable in cells lacking ATE1, a component of the N-End Rule pathway.

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An amino-acid position at the crossroads of evolution of protein function: antibiotic-sensor domain of the BlaR1 protein from Staphylococcus aureus vs. class D beta-lactamases.

Publication Date: 2012 Jan 18 PMID: 22262858
Authors: Kumarasiri, M. - Llarrull, L. I. - Borbulevych, O. - Fishovitz, J. - Lastochkin, E. - Baker, B. M. - Mobashery, S.
Journal: J Biol Chem

The integral-membrane protein BlaR1 of Staphylococcus aureus senses the presence of beta-lactam antibiotics in the milieu, transduces the information to its cytoplasmic side, where its activity unleashes the expression of a set of genes, including that for BlaR1 itself, which manifest the antibiotic-resistant phenotype. The x-ray structure of the sensor domain of this protein exhibits an uncanny similarity to those of the class D beta-lactamases. The former is a membrane-bound receptor/sensor for the beta-lactam antibiotics, devoid of catalytic competence for substrate turnover, whereas the latter are soluble periplasmic enzymes in Gram-negative bacteria with avid ability for beta-lactam turnover. The two are clearly related to each other from an evolutionary point of view. However, the high-resolution x-ray structures for both by themselves do not reveal why one is a receptor and the other an enzyme. It is documented herein that a single amino-acid change at position 439 of the BlaR1 protein is sufficient to endow the receptor/sensor protein with modest turnover ability for cephalosporins as substrates. The x-ray structure for this mutant protein and the dynamics simulations revealed how a hydrolytic water molecule may sequester itself in the antibiotic-binding site to enable hydrolysis of the acylated species. These studies document how the nature of the residue at position 439 is critical for the fate of the protein in imparting unique functions on the same molecular template, to result in one as a receptor and in another as a catalyst.

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Loss of the lysosomal ion channel TRPML1 leads to cathepsin B-dependent apoptosis.

Publication Date: 2012 Jan 18 PMID: 22262857
Authors: Colletti, G. A. - Miedel, M. T. - Quinn, J. - Andharia, N. - Weisz, O. A. - Kiselyov, K.
Journal: J Biol Chem

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases. Here we report that an acute siRNA mediated loss of TRPML1 specifically causes a leak of lysosomal protease Cathepsin B (CatB) into the cytoplasm. CatB leak is associated with apoptosis, which can be prevented by CatB inhibition. Inhibition of proapoptotic protein Bax prevents TRPML1 KD mediated apoptosis but does not prevent cytosolic release of CatB. This is the first evidence providing mechanistic link between acute TRPML1 loss and cell death.

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Functional and structural characterization of an alpha-(1->2) branching sucrase derived from DSR-E glucansucrase.

Publication Date: 2012 Jan 18 PMID: 22262856
Authors: Brison, Y. - Pijning, T. - Malbert, Y. - Fabre, E. - Mourey, L. - Morel, S. - Potocki-Veronese, G. - Monsan, P. - Tranier, S. - Remaud-Simeon, M. - Dijkstra, B. W.
Journal: J Biol Chem

DeltaN123-GBD-CD2 is a truncated form of the bi-functional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbours the second catalytic domain of DSR-E. Like GBD-CD2 this variant displays alpha-(1-->2)branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of alpha-(1-->2)linkages. The crystal structure of the apo alpha-(1-->2)branching sucrase DeltaN123-GBD-CD2 was solved at 1.90 A resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-DeltaN and GTF-SI glucansucrases of glycoside hydrolase family 70 (GH70). Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-DeltaN and DeltaN123-GBD-CD2. Subsite +1 analysis revealed three residues (A2249, G2250 and F2214) that are specific to DeltaN123-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-DeltaN showed that A2249 and G2250 are not directly involved in substrate binding and regiospecificity. In constrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to DeltaN123-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of alpha-(1-->2)branches.

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Met kinase-dependent loss of the E3 ligase Cbl in gastric cancer.

Publication Date: 2012 Jan 18 PMID: 22262855
Authors: Lai, A. Z. - Durrant, M. - Zuo, D. - Ratcliffe, C. D. - Park, M.
Journal: J Biol Chem

Strict regulation of signaling by receptor tyrosine kinases (RTKs) is essential for normal biological processes, and disruption of this regulation can lead to tumor initiation and progression. Signal duration by the Met RTK is mediated in part by the E3 ligase Cbl. Cbl is recruited to Met upon kinase activation and promotes ubiquitination, trafficking, and degradation of the receptor. The Met RTK has been demonstrated to play a role in various types of cancer. Here, we show that Met-dependent loss of Cbl protein in MET-amplified gastric cancer cell lines represents another mechanism contributing to signal dysregulation. Loss of Cbl protein is dependent on Met kinase activity and is partially rescued with a proteasome inhibitor, lactacystin. Moreover, Cbl loss not only uncouples Met from Cbl-mediated negative regulation, but also releases other Cbl-targets, such as the EGF receptor, from Cbl-mediated signal attenuation. Thus, Met-dependent Cbl loss may also promote cross-talk through indirect enhancement of EGFR signaling.

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A Three-dimensional Structure of Steroid 21-Hydroxylase (Cytochrome P450 21A2) with Two Substrates Reveals Locations of Disease-associated Variants.

Publication Date: 2012 Jan 18 PMID: 22262854
Authors: Zhao, B. - Lei, L. - Kagawa, N. - Sundaramoorthy, M. - Banerjee, S. - Nagy, L. D. - Guengerich, F. P. - Waterman, M. R.
Journal: J Biol Chem

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ~95% of individuals with congenital adrenal hyperplasia (CAH), a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 A resolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme; the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features not only locate the key substrate recognition residues around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of CAH. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.

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The Latency-Associated Peptide of Transforming Growth Factor Beta is Not Subject to Physiological Mannose Phosphorylation.

Publication Date: 2012 Jan 18 PMID: 22262853
Authors: Barnes, J. - Warejcka, D. - Simpliciano, J. - Twining, S. - Steet, R.
Journal: J Biol Chem

Latent TGF-beta1 was one of the first non-lysosomal glycoproteins reported to bear mannose 6-phosphate (Man-6-P) residues on its N-glycans. Prior studies have suggested that this sugar modification regulates the activation of latent TGF-beta1 by allowing it to bind cell surface-localized Man-6-P receptors. Man-6-P has also been proposed as an anti-scarring therapy based on its ability to directly block the activation of latent TGF-beta1. A complete understanding of the physiological relevance of latent TGF-beta1 mannose phosphorylation, however, is still lacking. Here we investigate the degree of mannose phosphorylation on secreted latent TGF-beta1 and examine its Man-6-P dependent activation in primary human corneal stromal fibroblasts. Contrary to earlier reports, minimal to no Man-6-P modification was found on secreted and cell-associated latent TGF-beta1 produced from multiple primary and transformed cell types. Results showed that the inability to detect Man-6-P residues was not due to masking by the latent TGF-beta1 binding protein (LTBP). Moreover, the efficient processing of glycans on latent TGF-beta1 to complex type structures was consistent with the lack of mannose phosphorylation during biosynthesis. We further demonstrated that the conversion of corneal stromal fibroblast to myofibroblasts, a well-known TGF-beta1 dependent process, was not altered by Man-6-P addition when latent forms of this growth factor were present. Collectively, these findings indicate that Man-6-P dependent effects on latent TGF-beta1 activation are not mediated by direct modification of its latency-associated peptide.

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Characterization of BRCA1 centrosome targeting, dynamics and function: A role for the nuclear export signal, CRM1 and Aurora A kinase.

Publication Date: 2012 Jan 18 PMID: 22262852
Authors: Brodie, K. M. - Henderson, B. R.
Journal: J Biol Chem

BRCA1 is a DNA damage response protein and functions in the nucleus to stimulate DNA repair and at the centrosome to inhibit centrosome overduplication in response to DNA damage. The loss or mutation of BRCA1 causes centrosome amplification and abnormal mitotic spindle assembly in breast cancer cells. The BRCA1/BARD1 heterodimer binds and ubiquitinates gamma-tubulin to inhibit centrosome amplification and promote microtubule nucleation, however regulation of BRCA1 targeting and function at the centrosome is poorly understood. Here we show that both N- and C-termini of BRCA1 are required for its centrosomal localization, and that BRCA1 moves to the centrosome independently of BARD1 and gamma-tubulin. Mutations in the C-terminal phosphoprotein-binding BRCT domain of BRCA1 prevented localization to centrosomes. Photobleaching experiments identified dynamic (60%) and immobilized (40%) pools of ectopic BRCA1 at the centrosome, and these are regulated by the nuclear export receptor CRM1 (chromosome region maintenance 1) and BARD1. CRM1 mediates nuclear export of BRCA1, and mutation of the export sequence blocked BRCA1 regulation of centrosome amplification in irradiated cells. CRM1 binds to undimerized BRCA1 and is displaced by BARD1. Photobleaching assays implicate CRM1 in driving undimerized BRCA1 to the centrosome and revealed that when BRCA1 subsequently binds to BARD1 it is less well retained at centrosomes, suggesting a mechanism to accelerate BRCA1 release after formation of the active heterodimer. Moreover, Aurora A binding and phosphorylation of BRCA1 enhanced its centrosomal retention and regulation of centrosome amplification. Thus, CRM1, BARD1 and Aurora A promote the targeting and function of BRCA1 at centrosomes.

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The substrate specificity of the two mitochondrial ornithine carriers can be swapped by a single mutation in the substrate binding site.

Publication Date: 2012 Jan 19 PMID: 22262851
Authors: Monne', M. - Miniero, D. V. - Daddabbo, L. - Robinson, A. J. - Kunji, E. R. - Palmieri, F.
Journal: J Biol Chem

Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, that share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that R179 and E180 of contact point II bind the Calpha carboxylate and amino group of the substrates, respectively. Residue E77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because R179 is probably connected with R275 of contact point III through W224 by cation-pi interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that R179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.

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PREPARATION OF A SITE-SPECIFIC T=mCG CIS-SYN CYCLOBUTANE DIMER-CONTAINING TEMPLATE AND ITS ERROR-FREE BYPASS BY YEAST AND HUMAN POLYMERASE ETA.

Publication Date: 2012 Jan 18 PMID: 22262850
Authors: Song, Q. - Sherrer, S. M. - Suo, Z. - Taylor, J. S.
Journal: J Biol Chem

C to T mutations are a hallmark of UV light, and in humans occur at preferentially at methylated Py(m)CG sites, which are also sites of preferential cyclobutane pyrimidine dimer (CPD) formation. In response, cells have evolved DNA damage bypass polymerases, of which, polymerase eta (Poleta) appears to be specifically adapted to synthesize past cis-syn cyclobutane dimers (CPD). While T=T CPDs are stable, CPDs containing C or 5-methylC ((m)C) are not, and spontaneously deaminate to U or T at pH 7 and 37 degrees C over a period of hours or days, making their preparation and study difficult. Furthermore, there is evidence to suggest that depending on solvent polarity a C or an (m)C in a CPD can adopt three tautomeric forms, one of which could code as a T. Although many in vitro studies have established that synthesis past T or U in a CPD by Poleta occurs in a highly error-free manner, the only in vitro evidence that synthesis past C or (m)C in a CPD also occurs in an error-free manner is for an (m)C in the 5'-position of a (m)C=T CPD. Herein, we describe the preparation and characterization of an ODN containing a CPD of a T(m)CG site, one of the major sites of C methylation and C to T mutations found in the p53 gene of basal and squamous cell cancers. We also demonstrate that both yeast and human Poleta synthesize past the 3'-(m)C CPD in a >99% error-free manner, consistent with the highly water exposed nature of the active site.

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The leukemia-associated fusion-protein Tel-platelet derived growth factor receptor B (Tel-PdgfRB) inhibits transcriptional repression of the PTPN13 gene by the interferon consensus sequence binding protein (Icsbp).

Publication Date: 2012 Jan 18 PMID: 22262849
Authors: Huang, W. - Hu, L. - Bei, L. - Hjort, E. - Eklund, E. A.
Journal: J Biol Chem

Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. In previous studies, we identified the gene encoding Fas-associated phosphatase 1 (Fap1; the PTPN13 gene) as an Icsbp target. In the current study, we determine that repression of PTPN13 by Icsbp requires cooperation with Tel and histone deacetylase 3 (Hdac3). These factors form a multi-protein complex which requires pre-binding of Tel to the PTPN13 cis element with subsequent recruitment of Icsbp and Hdac3. We found that knockdown of Tel or Hdac3 in myeloid cells increases Fap1 expression and results in Fap1-dependent resistance to Fas-induced apoptosis. The TEL gene was initially identified due to involvement in leukemia-associated chromosomal translocations. The first identified TEL translocation partner was the gene encoding Platelet Derived Growth Factor receptor B (PdgfRB). The resulting Tel-PdgfRB fusion protein exhibits constitutive tyrosine kinase activity and influences cellular proliferation. In the current studies, we find that Tel-PdgfRB influences apoptosis in a manner that is independent of tyrosine kinase activity. We found that Tel-PdgfRB expressing myeloid cells have increased Fap1 expression and Fap1-dependent Fas-resistance. We determined that interaction between Tel and Tel-PdgfRB decreases Tel/Icsbp/Hdac3 binding to the PTPN13 cis element, resulting in increased transcription. Therefore, these studies identify a novel mechanism by which the Tel-PdgfRB oncoprotein may contribute to leukemogenesis.

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The role of Ras guanine nucleotide releasing protein 4 in Fc{varepsilon}RI-mediated signaling, mast cell function, and T cell development.

Publication Date: 2012 Jan 19 PMID: 22262848
Authors: Zhu, M. - Fuller, D. M. - Zhang, W.
Journal: J Biol Chem

The RasGRP (Ras guanine nucleotide releasing protein) family proteins are guanine nucleotide exchange factors that activate Ras GTPases, ultimately leading to MAPK activation and many cellular processes. The RasGRP family has four members. Published studies demonstrate that RasGRP1, RasGRP2, and RasGRP3 play critical roles in T cells, platelets, and B cells, respectively. RasGRP4 is highly expressed in mast cells. While previous data suggest that it is important in mast cell development and function, the role of RasGRP4 in mast cells and allergic responses has not been clearly demonstrated. In this study, we generated RasGRP4-/- mice to examine the function of RasGRP4. Analyses of these mice showed that mast cells were able to develop normally in vivo and in vitro. Despite high levels of RasGRP4 expression in mast cells, RasGRP4 deficiency led to only a modest reduction in FcepsilonRI-mediated degranulation and cytokine production. Interestingly, mast cells deficient in both RasGRP1 and RasGRP4 had a much more severe block in FcepsilonRI-mediated signaling and mast cell function. We also made the unexpected finding that RasGRP4 functions during thymocyte development. Our data suggest that after the engagement of immunoreceptors, immune cells likely employ multiple members of the RasGRP family to transduce critical signals.

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Serine/threonine phosphatase (SP-STP), secreted from Streptococcus pyogenes, is a proapoptotic protein.

Publication Date: 2012 Jan 19 PMID: 22262847
Authors: Agarwal, S. - Agarwal, S. - Jin, H. - Pancholi, P. - Pancholi, V.
Journal: J Biol Chem

The present investigation illustrates an important property of eukaryote-type serine/threonine phosphatase (SP-STP) of Group A Streptococcus (GAS) in causing programmed cell death of human pharyngeal cells. The secretory nature of SP-STP, its elevated expression in the intracellular GAS and the ability of wild-type GAS but not the GAS mutant devoid of SP-STP to cause apoptosis of the host cell both in vitro and in vivo, suggest that GAS deploys SP-STP as an important virulence determinant to exploit host cell machinery for its own advantage during infection. The exogenously added SP-STP is able to enter the cytoplasm, and subsequently traverses into the nucleus in a temporal fashion to cause apoptosis of the pharyngeal cells. The programmed cell death induced by SP-STP, which requires active transcription and de novo protein synthesis, is also caspase-dependent. Further, the entry of SP-STP into the cytoplasm is dependent on its secondary structure as the catalytically inactive SP-STP with an altered structure is unable to internalize and cause apoptosis. The ectopically expressed wild-type SP-STP was found to be in the nucleus and conferred apoptosis of Detroit 562 pharyngeal cells. However, the catalytically inactive SP-STP was incompetent to cause apoptosis even when intracellularly expressed. The ability of SP-STP to activate pro-apoptotic signaling cascades both in the cytoplasm and in the nucleus resulted into mitochondrial dysfunctioning and perturbation in the phosphorylation status of histones in the nucleus. SP-STP thus not only functions as a virulence regulator, but also as an important factor responsible for host-related pathogenesis.

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Recognition of human tumor necrosis factor TNF-alpha by a therapeutic antibody fragment: energetics and structural features.

Publication Date: 2012 Jan 19 PMID: 22262846
Authors: Marusi, C. J - Podlipnik, C. - Jevsevar, S. - Kuzman, D. - Vesnaver, G. - Lah, J.
Journal: J Biol Chem

Human tumor necrosis factor alpha (TNF-alpha) exists in its functional state as a homotrimeric protein and is involved in inflammation processes and immune response of a human organism. Overproduction of TNF-alpha results in development of chronic autoimmune diseases that can be successfully treated by inhibitors such as monoclonal antibodies. However, the nature of antibody-TNF-alpha recognition remains elusive due to insufficient understanding of its molecular driving forces. Therefore, we studied energetics of binding of a therapeutic antibody fragment (Fab) to the native and non-native forms of TNF-alpha by employing calorimetric and spectroscopic methods. Global thermodynamic analysis of data obtained from the corresponding binding and urea-induced denaturation experiments has been supported by structural modeling. We demonstrate that the observed high affinity binding of Fab to TNF-alpha is an enthalpy driven process due mainly to specific non-covalent interactions taking place at the Fab-TNF-alpha binding interface. It is coupled to entropically unfavorable conformational changes and accompanied by favorable solvation contributions. Moreover, the three-state model analysis of TNF-alpha unfolding shows that at physiological concentrations TNF-alpha may exist not only as a biologically active trimer but also as an inactive monomer. It further suggests that even small changes of TNF-alpha concentration could have a considerable effect on the TNF-alpha activity. We believe that this study sets the energetic basis for understanding of TNF-alpha inhibition by antibodies and its unfolding linked with the concentration dependent activity regulation.

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The T-cell receptor is a structure capable of initiating signalling in the absence of large conformational rearrangements.

Publication Date: 2012 Jan 19 PMID: 22262845
Authors: Fernandes, R. A. - Shore, D. A. - Vuong, M. T. - Yu, C. - Zhu, X. - Pereira-Lopes, S. - Brouwer, H. - Fennelly, J. A. - Jessup, C. M. - Evans, E. J. - Wilson, I. A. - Davis, S. J.
Journal: J Biol Chem

Native and non-native ligands of the TCR, including antibodies, have been proposed to induce signalling in T-cells via intra- or inter-subunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3epsilon complexed with the mitogenic anti-CD3epsilon antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3epsilon from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3epsilon. Saturation mutagenesis of surface-exposed CD3epsilon residues, coupled with assays of antibody-induced signalling by the mutated complexes, suggest a new configuration for the complex within which CD3epsilon is highly exposed, and reveals that no large new CD3epsilon interfaces are required to form during antibody-induced signalling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signalling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signalling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.

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Ubiquitin-specific protease 4 mitigates Toll-like/interleukin-1 receptor signaling and regulates innate immune activation.

Publication Date: 2012 Jan 19 PMID: 22262844
Authors: Zhou, F. - Zhang, X. - Dam, H. V. - Dijke, P. T. - Huang, H. - Zhang, L.
Journal: J Biol Chem

The Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) signaling pathway is essential for innate immune responses and immune homeostasis. Lysine 63 polyubiquitinated Tumor necrosis factor receptor-associated factor (TRAF)6 mediates its downstream signaling activation. In a gain of expression screen of 66 different deubiquitinating enzymes (DUBs), we identified ubiquitin-specific protease (USP)4 as a potent negative regulator of TLR/IL-1R signaling and TRAF6 interacting protein. USP4 deubiquitinates TRAF6 and thereby prevents the activation of NF-kappaB and AP-1 transcriptional factors and subsequent pro-inflammatory responses. LPS-treated usp4-depleted zebrafish larvaes expressed higher levels of pro-inflammatory cytokines and were more susceptible to endotoxic challenge. Taken together, our results demonstrate that USP4 plays an essential role in negative regulation of the TLR-IL-1R signaling-mediated innate immune response.

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High-affinity neurexin binding to the cell-adhesion G-protein coupled receptor CIRL1/Latrophilin-1 produces an intercellular adhesion complex.

Publication Date: 2012 Jan 19 PMID: 22262843
Authors: Boucard, A. A. - Ko, J. - Sudhof, T. C.
Journal: J Biol Chem

The G-protein coupled receptor CIRL1/Latrophilin-1 (abbreviated as CL1) and the type-1 membrane neurexin proteins represent distinct neuronal cell-adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for alpha-latrotoxin, a component of black-widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we now identify a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice-site #4. Using saturation binding assays, we show that neurexins lacking an insert at SS#4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS#4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well-characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/ threonine-rich, hormone-binding, and GAIN domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell-adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction is trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well-characterized interaction with intracellular SHANK adaptor proteins, and could form a trans-synaptic complex with presynaptic neurexins.

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Constitutive clathrin-mediated endocytosis of CTLA-4 persists during T cell activation.

Publication Date: 2012 Jan 20 PMID: 22262842
Authors: Qureshi, O. S. - Kaur, S. - Hou, T. - Jeffery, L. E. - Poulter, N. S. - Briggs, Z. - Kenefeck, R. - Willox, A. K. - Royle, S. J. - Rappoport, J. Z. - Sansom, D. M.
Journal: J Biol Chem

CTLA-4 is one of the most important negative regulators of the T cell immune response. However, the subcellular distribution of CTLA-4 is unusual for a receptor that interacts with cell surface trans-membrane ligands in that CTLA-4 is rapidly internalised from the plasma membrane. It has been proposed that T cell activation can lead to stabilization of CTLA-4 expression at the cell surface. Here we have analysed in detail the internalisation, recycling and degradation of CTLA-4. We demonstrate that CTLA-4 is rapidly internalised from the plasma membrane in a clathrin and dynamin dependent manner driven by the well- characterised YVKM trafficking motif. Furthermore, we show that once internalised, CTLA-4 co-localises with markers of recycling endosomes and is recycled to the plasma membrane. Whilst we observed limited co-localisation of CTLA-4 with lysosomal markers, CTLA-4 was nonetheless degraded in a manner inhibited by lysosomal blockade. T cell activation stimulated mobilization of CTLA-4, as judged by an increase in cell surface expression, however this pool of CTLA-4 continued to endocytose and was not stably retained at the cell surface. These data support a model of trafficking whereby CTLA-4 is constitutively internalised in a ligand-independent manner undergoing both recycling and degradation. Stimulation of T cells increases CTLA-4 turnover at the plasma membrane, however CTLA-4 endocytosis continues and is not stabilised during activation of human T cells. These findings emphasize the importance of clathrin-mediated endocytosis in regulating CTLA-4 trafficking throughout T cell activation.

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Mitochondrial 3-beta hydroxysteroid dehydrogenase enzyme activity requires a reversible pH-dependent conformational change at the intermembrane space.

Publication Date: 2012 Jan 19 PMID: 22262841
Authors: Prasad, M. - Thomas, J. L. - Whittal, R. M. - Bose, H. S.
Journal: J Biol Chem

The inner mitochondrial membrane protein 3-beta hydroxysteroid dehydrogenase2 (3betaHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3betaHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3betaHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3betaHSD2 retained its primarily alpha-helical character with a decrease in alpha-helical content at lower pHs, while the beta-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 minutes. Metabolic conversion assays indicated peak 3betaHSD2 activity at pH 4.5 with approximately 2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing 3betaHSD2 concentration from 1mug to 40 mug resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with Guanidinum Hydrochloride (GnHCl) showed a three-step cooperative unfolding of 3betaHSD2 from pH 7.4 to pH 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GnHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4 to 5, 3betaHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.

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Distinct control of MYD88-dependent and AKT-regulated responses by the IL-1RI co-receptor, TILRR.

Publication Date: 2012 Jan 19 PMID: 22262840
Authors: Zhang, X. - Montagut Pino, G. - Shephard, F. - Kiss-Toth, E. - Qwarnstrom, E. E.
Journal: J Biol Chem

Inflammatory responses are controlled through members of the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) super family. Our earlier work demonstrates that the IL-1 receptor type 1 (IL-1RI) co-receptor, TILRR, amplifies IL-1 activation of NF-kappaB and inflammatory genes. Here we show that TILRR similarly promotes IL-1-induced anti-apoptotic signals and reduces Caspase-3 activity. Further, the TILRR induced effects on cell survival and inflammatory responses are controlled through distinct parts of the IL-1RI regulatory TIR domain. Alanine scanning mutagenesis identified a functional TILRR mutant (R425A), which blocked increases in cell survival and upstream activation of Akt, but had no effect on amplification of MyD88- dependent inflammatory responses. A second mutant (D448A) blocked TILRR potentiation of MyD88-dependent signals and inflammatory activation but had no impact on cell survival. Secondary structure predictions suggested that the mutations induce distinct alterations in the alpha-helical structure of the TILRR core protein. The results indicate a role for TILRR in selective amplification of NF-kappaB responses through IL-1RI and suggest that the specificity is determined by changes in receptor conformation and adapter protein recruitment.

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Essential roles of Raf/ERK/MAPK pathway, YY1, and Ca+2 influx in the growth arrest of human vascular smooth muscle cells by bilirubin.

Publication Date: 2012 Jan 18 PMID: 22262839
Authors: Stoeckius, M. - Erat, A. - Fujikawa, T. - Hiromura, M. - Koulova, A. - Otterbein, L. - Bianchi, C. - Tobiasch, E. - Dagon, Y. - Sellke, F. W. - Usheva, A.
Journal: J Biol Chem

The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypo-phosphorylation on amino acids Ser608 and Ser780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum stimulated human vascular smooth muscle primary cell cultures (hVSMC) bilirubin favors growth arrest and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered Rb profile of hypo-phosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5S and 45S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.

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Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding.

Publication Date: 2012 Jan 17 PMID: 22262838
Authors: Storti, B. - Bizzarri, R. - Cardarelli, F. - Beltram, F.
Journal: J Biol Chem

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain-management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high-resolution/high-sensitivity fluorescence imaging techniques (such as FRET, Correlation Spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with RTX, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules, but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality towards activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling.

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Solution structure analysis of the cytoplasmic domain of podocyte protein NEPH1 using SWAXS (Small/Wide Angle X-ray Scattering).

Publication Date: 2012 Jan 17 PMID: 22262837
Authors: Mallik, L. - Arif, E. - Sharma, P. - Rathore, Y. S. - Wong, H. N. - Holzman, L. B. - Ashish, A. - Nihalani, D.
Journal: J Biol Chem

Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by SWAXS (small/wide angle X-ray scattering). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 A, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD-ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues THV, residues K761 and Y762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into Neph1-CD molecular structure and function.

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The Get5 carboxyl terminal domain is a novel dimerization motif that tethers an extended Get4/Get5 complex.

Publication Date: 2012 Jan 17 PMID: 22262836
Authors: Chartron, J. W. - Vandervelde, D. G. - Rao, M. - Clemons, W. M. Jr
Journal: J Biol Chem

Tail-anchored (TA) transmembrane proteins are targeted to membranes post- translationally. The proteins Get4 and Get5 form an obligate complex that catalyzes the transfer of TA proteins destined to the endoplasmic reticulum (ER) from Sgt2 to the cytosolic targeting factor Get3. Get5 forms a homodimer mediated by its carboxyl domain. We show here that a conserved motif exists within the carboxyl domain. A high-resolution crystal structure and solution NMR structures of this motif reveal a novel and stable helical dimerization domain. We additionally determined a solution NMR structure of a divergent fungal homolog and comparison of these structures allows annotation of specific stabilizing interactions. Using solution X-ray scattering and the structures of all folded domains, we present a model of the full length Get4/Get5 complex.

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Nitrogen Monoxide (NO) Storage and Transport by Dinitrosyl-Dithiol-Iron Complexes: Long-Lived NO that is Trafficked by Interacting Proteins.

Publication Date: 2012 Jan 19 PMID: 22262835
Authors: Suryo Rahmanto, Y. - Kalinowski, D. S. - Lane, D. J. - Lok, H. C. - Richardson, V. - Richardson, D. R.
Journal: J Biol Chem

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Structure of the C-terminal tandem BRCT repeats of Rtt107 reveals a critical role in the interaction with gammaH2A during DNA damage repair.

Publication Date: 2012 Jan 19 PMID: 22262834
Authors: Li, X. - Liu, K. - Li, F. - Wang, J. - Huang, H. - Wu, J. - Shi, Y.
Journal: J Biol Chem

Rtt107 (regulator of Ty1 transposition 107, or Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55 and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between gammaH2A (phosphorylated histone H2A) and the C-terminal tandem BRCT repeats (BRCT5-BRCT6) of Rtt107. The crystal structures of BRCT5-BRCT6 alone and in a complex with gammaH2A reveal the molecular basis of the Rtt107-gammaH2A interaction. We used in vitro mutagenesis and a fluorescent polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrates the critical role of BRCT5-BRCT6 and its interaction with gammaH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.

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Neurodegeneration induced by the clustering of sialylated glycosylphosphatidylinositols of prion proteins.

Publication Date: 2012 Jan 23 PMID: 22262833
Authors: Bate, C. - Williams, A.
Journal: J Biol Chem

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrPSc). The mechanisms by which PrPSc accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons the addition of PrPSc alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A2 (cPLA2) and triggering synapse damage. These effects of PrPSc are dependent upon its glycosylphosphatidylinositol (GPI) anchor suggesting that it is the increased density of GPIs that occurs following the aggregation of PrPSc molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrPC) also increased membrane cholesterol, activated cPLA2 and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1 another GPI-anchored protein and were dependent on upon the GPI anchor attached to PrPC containing two acyl chains and sialic acid. We propose that the aggregation of PrPSc, or the cross-linkage of PrPC, causes the clustering of sialic acid-containing GPI anchors at high densities resulting in altered membrane composition, the pathological activation of cPLA2 and synapse damage.

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Induction of ZEB by inactivation of RB is a key determinant of the mesenchymal phenotype of breast cancer.

Publication Date: 2012 Jan 19 PMID: 22262832
Authors: Arima, Y. - Hayashi, H. - Sasaki, M. - Hosonaga, M. - Goto, T. M. - Chiyoda, T. - Kuninaka, S. - Shibata, T. - Ohata, H. - Nakagama, H. - Taya, Y. - Saya, H.
Journal: J Biol Chem

We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition (EMT). To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation as well as induced epithelial marker expression. These results implicate ZEB in induction of the EMT as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase (CDK) inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

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The endoplasmic reticulum stress transducer OASIS is involved in the terminal differentiation of goblet cells in the large intestine.

Publication Date: 2012 Jan 19 PMID: 22262831
Authors: Asada, R. - Saito, A. - Kawasaki, N. - Kanemoto, S. - Iwamoto, H. - Oki, M. - Miyagi, H. - Izumi, S. - Imaizumi, K.
Journal: J Biol Chem

OASIS is a bZIP transmembrane transcription factor localized in the endoplasmic reticulum (ER) that is cleaved in its transmembrane region in response to ER stress. This novel ER stress transducer has been demonstrated to express in osteoblasts and astrocytes and promote terminal maturation of these cells. Additionally, OASIS is highly expressed in goblet cells of the large intestine. In this study, we investigated the roles of OASIS in goblet cell differentiation in the large intestine. To analyze the functions of OASIS in goblet cells, we examined morphological changes and the expression of goblet cell differentiation markers in the large intestine of Oasis-/- mice. By disrupting the Oasis gene, the number of goblet cells and production of mucus were decreased in the large intestine. Oasis-/- goblet cells showed abnormal morphology of mucous vesicles and rough ER. The expression levels of mature goblet cell markers were lower, and conversely those of early goblet cell markers were higher in Oasis-/- mice, indicating that differentiation from early to mature goblet cells is impaired in Oasis-/- mice. To determine the association of OASIS with other factors involved in goblet cell differentiation, in vitro experiments using cell culture model were performed. We found that OASIS was activated in response to mild ER stress which is induced in differentiating goblet cells. Knockdown of the Oasis transcript perturbed goblet cell terminal differentiation. Together, our data indicate that OASIS plays crucial roles in promoting the differentiation of early goblet cells to mature goblet cells in the large intestine.

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RACK1 promotes non-small-cell lung cancer tumorigenicity through activating sonic hedgehog signaling pathway.

Publication Date: 2012 Jan 19 PMID: 22262830
Authors: Shi, S. - Deng, Y. Z. - Zhao, J. S. - Ji, X. D. - Shi, J. - Feng, Y. X. - Li, G. - Li, J. J. - Zhu, D. - Koeffler, H. P. - Zhao, Y. - Xie, D.
Journal: J Biol Chem

Non-small-cell lung cancer (NSCLC) is a deadly disease due to lack of effective diagnosis biomarker and therapeutic target. Much effort has been made in defining gene defects in NSCLC, but its full molecular pathogenesis remains unexplored. Here, we found RACK1 (Receptor of Activated Kinase 1) was elevated in most NSCLC, and its expression level correlated with key pathological characteristics including tumor differentiation, stage and metastasis. In addition, RACK1 activated sonic hedgehog signaling pathway by interacting with and activating Smoothened to mediate Gli1-dependent transcription in NSCLC cells. And silencing RACK1 dramatically inhibited in vivo tumor growth and metastasis by blocking the sonic hedgehog signaling pathway. These results suggest that RACK1 represents a new promising diagnosis biomarker and therapeutic target for NSCLC.

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The Role of Runt-Related Transcription Factor 3 (RUNX3) in Transcription Regulation of Natural Cytotoxicity Receptor 1 (NCR1/NKp46), an Activating NK Cell Receptor.

Publication Date: 2012 Jan 17 PMID: 22253448
Authors: Lai, C. B. - Mager, D. L.
Journal: J Biol Chem

Natural cytotoxicity receptor 1 (NCR1), also known as NKp46, is a natural killer (NK) lymphocyte activating receptor. It is involved in major aspects of NK immune function and shows a high degree of lineage specificity in blood and bone marrow. The nature of its NK restricted expression is not well understood. In this study, we confirm that human NCR1 NKspecific expression is achieved at the mRNA level. We found two key cis-regulatory elements in the immediate vicinity upstream of the gene. One element acts as an essential promoter while the other a tissue-dependent enhancer/repressor. This latter regulatory element contains a runt related-transcription factor (RUNX) recognition motif that preferentially binds RUNX3. Interfering with RUNX proteins using a dominant negative form results in decreased Ncr1 expression. RUNX3 overexpression had the opposite effect. These findings shed light on the role of RUNX3 in the control of an important NK activating receptor.

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Molecular Determinants of Proteolytic Disassembly of the Reovirus Outer Capsid.

Publication Date: 2012 Jan 17 PMID: 22253447
Authors: Doyle, J. D. - Danthi, P. - Kendall, E. A. - Ooms, L. S. - Wetzel, J. D. - Dermody, T. S.
Journal: J Biol Chem

Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by subsequent viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer-capsid protein sigma3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) sigma3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The sigma3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable to T3D. We hypothesize that one or more additional sigma3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A sigma3 polymorphisms introduced individually into T3D-sigma3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of sigma3 proteolytic susceptibility. Variants with Y354H in sigma3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control sigma3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.

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Phosphorylation of Calcineurin B-like (CBL) calcium sensor proteins by their CBL-interacting protein kinases (CIPKs) is required for full activity of CBL-CIPK complexes towards their target proteins.

Publication Date: 2012 Jan 17 PMID: 22253446
Authors: Hashimoto, K. - Eckert, C. - Anschuetz, U. - Scholz, M. - Held, K. - Waadt, R. - Reyer, A. - Hippler, M. - Becker, D. - Kudla, J.
Journal: J Biol Chem

Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine-threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C-terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation occurs strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K(+) channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23 and AKT1 we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity towards its substrate. Together these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C-terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes towards their target proteins.

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Phosphatidylinositol 4-Kinase III beta is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry.

Publication Date: 2012 Jan 17 PMID: 22253445
Authors: Yang, N. - Ma, P. - Lang, J. - Zhang, Y. - Deng, J. - Ju, X. - Zhang, G. - Jiang, C.
Journal: J Biol Chem

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase III beta (PI4KB) is required for cellular entry by pseudoviruses bearing the SARS-CoV spike protein and that the cell entry mediated by SARS-CoV S is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV S-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization, but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection.

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Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD.

Publication Date: 2012 Jan 17 PMID: 22253444
Authors: Iglesias-Guimarais, V. - Gil-Guinon, E. - Gabernet, G. - Garcia-Belinchon, M. - Sanchez-Osuna, M. - Casanelles, E. - Comella, J. X. - Yuste, V. J.
Journal: J Biol Chem

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable to that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed and dff40/cad cDNA sequence does not reveal mutations altering its aminoacidic composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells, DFF40/CAD cytosolic levels can be restored by the over-expression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosomal-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinant in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.

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Loss of brain-enriched miR-124 enhances the stem-like traits and invasiveness of glioma cells.

Publication Date: 2012 Jan 17 PMID: 22253443
Authors: Xia, H. - Cheung, W. K. - Ng, S. S. - Jiang, X. - Jiang, S. - Sze, J. - Leung, G. K. - Gang, L. - Chan, D. T. - Bian, X. W. - Kung, H. F. - Poon, W. S. - Lin, M. C.
Journal: J Biol Chem

miR-124 is a brain-enriched miRNA that plays crucial role in neural development and has been shown to be downregulated in glioma and medulloblastoma, suggesting its possible involvement in brain tumor progression. Here we show that miR-124 is down-regulated in a panel of different grades of glioma tissues and all of the human glioma cell lines we examined. By integrated bioinformatics analysis and experimental confirmation, we identified SNAI2, which is often upregulated in glioma, as a direct functional target of miR-124. Since SNAI2 has been shown to regulate stem cell functions, we examined the roles of miR-124 and SNAI2 in glioma stem-like cell traits. The results showed that overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133+ cell subpopulation and stem cell markers (BMI1, Nanog and Nestin) expressions, and these effects could be rescued by re-expression of SNAI2. Furthermore, enhanced miR-124 expression significantly inhibited glioma cell invasion in vitro. Finally, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo. These findings reveal for the first time the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2.

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Solution structure of the HR domain of the type II secretion system.

Publication Date: 2012 Jan 17 PMID: 22253442
Authors: Gu, S. - Kelly, G. - Wang, X. - Frenkiel, T. - Shevchik, V. E. - Pickersgill, R. W.
Journal: J Biol Chem

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 core proteins mostly multimeric which build a sophisticated secretion machine spanning both bacterial membranes. OutC (GspC) is the core component of the inner-membrane sub-complex, thought to be involved in both recognition of substrate and interaction with the outer-membrane secretin, OutD (GspD). Here we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a beta-sandwich-like fold consisting of two beta-sheets each composed of three anti-parallel beta-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the T2SS have a structural or sequence ortholog within the T4PS. The HR domain is shown to interact with the N0 domain of the secretin such that beta1 of HR interacts with beta3 of N0. The importance of this interaction is explored in the context of the functional secretion system.

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Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition induces neurotoxicity via dysregulation of glutamate/calcium signaling and hyperexcitability.

Publication Date: 2012 Jan 17 PMID: 22253441
Authors: Ashpole, N. M. - Song, W. - Brustovetsky, T. - Engleman, E. A. - Brustovetsky, N. - Cummins, T. R. - Hudmon, A.
Journal: J Biol Chem

Aberrant glutamate and calcium signaling are neurotoxic to specific neuronal populations. Calcium/calmodulin-dependent kinase II (CaMKII), a multifunctional serine/threonine protein kinase in neurons, is believed to regulate neurotransmission and synaptic plasticity in response to calcium signaling produced by neuronal activity. Importantly, several CaMKII substrates control neuronal structure, excitability, and plasticity. Here, we demonstrate that CaMKII inhibition for >4 hours using small molecule and peptide inhibitors induces apoptosis in cultured cortical neurons. The neuronal death produced by prolonged CaMKII inhibition is associated with an increase in TUNEL staining, caspase-3 cleavage, and is blocked with translation inhibitor cycloheximide. Thus, this neurotoxicity is consistent with apoptotic mechanisms, a conclusion that is further supported by dysregulated calcium signaling with CaMKII inhibition. CaMKII inhibitory peptides also enhance the number of action potentials generated by a ramp depolarization, suggesting increased neuronal excitability with a loss of CaMKII activity. Extracellular glutamate concentrations are augmented with prolonged inhibition of CaMKII. Enzymatic buffering of extracellular glutamate and antagonism of the NMDA subtype of glutamate receptors prevent the calcium dysregulation and neurotoxicity associated with prolonged CaMKII inhibition. However, in the absence of CaMKII inhibition, elevated glutamate levels do not induce neurotoxicity, suggesting that a combination of CaMKII inhibition and elevated extracellular glutamate levels results in neuronal death. In sum, the loss of CaMKII observed with multiple pathological states in the central nervous system, including epilepsy, brain trauma, and ischemia, likely exacerbates programmed cell death by sensitizing vulnerable neuronal populations to excitotoxic glutamate signaling and inducing an excitotoxic insult itself.

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Z-Phe-Ala-diazomethylketone (PADK) disrupts and remodels the early oligomer states of the Alzheimer's disease Abeta42 protein.

Publication Date: 2012 Jan 17 PMID: 22253440
Authors: Zheng, X. - Gessel, M. M. - Wisniewski, M. L. - Viswanathan, K. - Wright, D. L. - Bahr, B. A. - Bowers, M. T.
Journal: J Biol Chem

The oligomerization of amyloid-beta protein (Abeta) is an important event in Alzheimer's disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Abeta and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Abeta42 form of the protein. The mass spectrum of a mixture of PADK and Abeta42 clearly shows that PADK binds directly to Abeta42 momomers and small oligomers. Ion mobility results indicate PADK not only inhibits the formation of Abeta42 dodecamers, but also removes pre-formed Abeta42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Abeta42 fibril formation in the solution. These results are consistent with a previous study which found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Abeta42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.

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Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions.

Publication Date: 2012 Jan 17 PMID: 22253439
Authors: Yin, L. M. - Edwards, M. A. - Li, J. - Yip, C. M. - Deber, C. M.
Journal: J Biol Chem

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms (including humans) and offer a viable alternative to conventional antibiotics as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In the present work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in tandem atomic force microscopy (AFM) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), circular dichroism spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane disruptive powers and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase of aggregated beta-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKK-ALFALWLAFLA-NH2) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKK-AAFAAWAAFAA-NH2). Re-distribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, while those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 muM, but the four-Leu peptides induced 40-80% hemolysis in the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (MICs = 4-32 muM). The overall findings suggest optimal routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.

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Biosynthesis, localisation and macromolecular arrangement of the Plasmodium falciparum translocon of exported proteins; PTEX.

Publication Date: 2012 Jan 17 PMID: 22253438
Authors: Bullen, H. E. - Charnaud, S. C. - Kalanon, M. - Riglar, D. T. - Dekiwadia, C. - Kangwanrangsan, N. - Torii, M. - Tsuboi, T. - Baum, J. - Ralph, S. A. - Cowman, A. F. - de Koning-Ward, T. F. - Crabb, B. S. - Gilson, P. R.
Journal: J Biol Chem

A major virulence determinant in the protozoan parasite Plasmodium falciparum is a protein complex known as PTEX (Plasmodium Translocon of EXported proteins). PTEX is a putative protein trafficking machinary responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex and in this study, three of the major stoichiometric components are investigated including; HSP101 (a AAA+ ATPase); a protein of no known function termed PTEX150; and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont-stage (PTEX150 and HSP101), or even earlier in the life-cycle (EXP2) and prior to invasion these components reside within the dense granules of invasive merozoites, From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230kDa which includes a ~600 kDa apparently homo-oligomeric complex of EXP2, that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components are arranged in the order of EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerises and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.

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An unusual N-terminal alphaalphabetaalphabetabetaalpha fold of PilQ from T. thermophilus mediates ring formation and is essential for piliation.

Publication Date: 2012 Jan 17 PMID: 22253437
Authors: Burkhardt, J. - Vonck, J. - Langer, J. D. - Salzer, R. - Averhoff, B.
Journal: J Biol Chem

DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual alphaalphabetaalphabetabetaalpha fold as a ring building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation and truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.

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Structural and molecular characterization of the iron-sensing hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5).

Publication Date: 2012 Jan 17 PMID: 22253436
Authors: Thompson, J. W. - Salahudeen, A. A. - Chollangi, S. - Ruiz, J. C. - Brautigam, C. A. - Makris, T. M. - Lipscomb, J. D. - Tomchick, D. R. - Bruick, R. K.
Journal: J Biol Chem

Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of Iron Regulatory Protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. FBXL5's own stability is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of FBXL5's N-terminus, a hemerythrin-like alpha-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.

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Mutual information analysis reveals coevolving residues in tat that compensate for two distinct functions in HIV-1 gene expression.

Publication Date: 2012 Jan 17 PMID: 22253435
Authors: Dey, S. S. - Xue, Y. - Joachimiak, M. P. - Friedland, G. D. - Burnett, J. C. - Zhou, Q. - Arkin, A. P. - Schaffer, D. V.
Journal: J Biol Chem

Viral genomes are continually subjected to mutations, and functionally deleterious ones can be rescued by reversion or additional mutations that restore fitness. The error prone nature of HIV-1 replication has resulted in highly diverse viral sequences, and it is not clear how viral proteins such as Tat, which plays a critical role in viral gene expression and replication, retain their complex functions. Although several important amino acid positions in Tat are conserved, we hypothesized that it may also harbor functionally important residues that may not be individually conserved yet appear as correlated pairs, whose analysis could yield new mechanistic insights into Tat function and evolution. To identify such sites, we combined Mutual Information analysis and experimentation to identify coevolving positions and found that residues 35 and 39 are strongly correlated. Mutation of either residue of this pair into amino acids that appear in numerous viral isolates yields a defective virus; however, simultaneous introduction of both mutations into the heterologous Tat sequence restores gene expression close to wild-type Tat. Furthermore, in contrast to most coevolving protein residues that contribute to the same function, structural modeling and biochemical studies showed that these two residues contribute to two mechanistically distinct steps in gene expression: binding P-TEFb and promoting P-TEFb phosphorylation of CTD in RNAPII. Moreover, Tat variants that mimic HIV-1 subtype B or C at sites 35 and 39 have evolved orthogonal strengths of P-TEFb binding vs. RNAPII phosphorylation, suggesting that subtypes have evolved alternate transcriptional strategies to achieve similar gene expression levels.

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Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis.

Publication Date: 2012 Jan 17 PMID: 22253434
Authors: Soga, N. - Kinosita, K. - Yoshida, M. - Suzuki, T.
Journal: J Biol Chem

The ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high-energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (DeltapH) across the membrane and transmembrane electrical potential (Deltapsi). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted and previous experimental results vary. Here we show that, in the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the epsilon subunit, DeltapH imposed by acid-base transition and Deltapsi produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis, within the experimental range examined (DeltapH -0.3 to 2.2, Deltapsi -30 to 140 mV, pH around the catalytic domain 8.0). Either DeltapH or Deltapsi alone can drive synthesis, even when the other slightly opposes. Deltapsi was estimated from the Nernst equation, which appeared valid down to 1 mM K(+) inside the proteoliposomes, thanks to careful removal of K(+) from the lipid.

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miR-30 family members negatively regulate osteoblast differentiation.

Publication Date: 2012 Jan 17 PMID: 22253433
Authors: Wu, T. - Zhou, H. - Hong, Y. - Li, J. - Jiang, X. - Huang, H.
Journal: J Biol Chem

miRNAs are endogenously expressed 18-25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to decrease of Alkaline phosphatase (ALP) activity, while knockdown of them increased the activity. Then bioinformatic analysis identi fi ed potential target sites of miR-30 family located in the 3'-untranslated regions (3'UTR) of Smad1 and Runx2. Western blot and qRT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression based on repression its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpression of Smad1 and Runx2 significantly eliminate the inhibitory effect of miR-30 on osteogenic differentiation, provided strong evidence that miR-30 mediate the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of miR-30 family were validated in mouse bone marrow mesenchymal stem cells (MSCs). Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.

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The Drosophila juvenile hormone receptor candidates Methoprene-tolerant (MET) and Germ cell-expressed (GCE) utilize a conserved LIxxL motif to bind the FTZ-F1 nuclear receptor.

Publication Date: 2012 Jan 16 PMID: 22249180
Authors: Bernardo, T. J. - Dubrovsky, E. B.
Journal: J Biol Chem

Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila S2 cells the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling we found that MET and GCE possess a C-terminal alpha-helix featuring a conserved motif LIxxL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1/MET and FTZ-F1/GCE heterodimer formation involves a typical NR box/AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.

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A new molecular bridge between RelA/p65 and NF-kappaB target genes via histone acetyltransferase TIP60.

Publication Date: 2012 Jan 16 PMID: 22249179
Authors: Kim, J. W. - Jang, S. M. - Kim, C. H. - An, J. H. - Kang, E. J. - Choi, K. H.
Journal: J Biol Chem

The nuclear factor-kappaB (NF-kappaB) family is involved in the expressions of numerous genes involved in development, apoptosis, inflammatory responses and oncogenesis. In this study, we identified four NF-kappaB target genes that are modulated by TIP60. We also found that TIP60 interacts with the NF-kappaB RelA/p65 subunit and increases its transcriptional activity through protein-protein interaction. Although TIP60 binds with RelA/p65 using its histone acetyltransferase domain, TIP60 does not directly acetylate RelA/p65. However, TIP60 maintained acetylated K310 RelA/p65 levels in TNF-alpha dependent NF-kappaB signaling pathway. In chromatin immunoprecipitation assay, TIP60 was primarily recruited to the IL-6, IL-8, C-IAP1 and XIAP promoters in TNF-alpha stimulation, followed by acetylation of histones H3 and H4. Chromatin remodeling by TIP60 involved the sequential recruitment of acetyl-K310 RelA/p65 to its target gene promoters. Furthermore, we showed that up-regulated TIP60 expression was correlated with acetyl-K310 RelA/p65 expressions in hepatocarcinoma tissues. Taken together, these results suggest that TIP60 is involved in the NF-kappaB pathway through protein interaction with RelA/p65 and that it modulates the transcriptional activity of RelA/p65 in NF-kappaB dependent gene expression.

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Structural analysis of the plasmodium falciparum Erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope.

Publication Date: 2012 Jan 16 PMID: 22249178
Authors: Mayer, C. - Slater, L. - Erat, M. C. - Konrat, R. - Vakonakis, I.
Journal: J Biol Chem

P. falciparum infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS centre is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

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Role of Cdc14 in Timing the End of Cell Division{diamondsuit}: Cdc14 Phosphatases Preferentially Dephosphorylate a Subset of Cyclin-dependent Kinase (Cdk) Sites Containing Phosphoserine.

Publication Date: 2012 Jan 13 PMID: 22247558
Authors:
Journal: J Biol Chem

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Regulation of insulin signalling and glucose transporter 4 (GLUT4) exocytosis by the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase, SKIP.

Publication Date: 2012 Jan 15 PMID: 22247557
Authors: Ijuin, T. - Takenawa, T.
Journal: J Biol Chem

The glucose transporter 4 (GLUT4) is responsible for glucose uptake in the skeletal muscle. Insulin-induced translocation of GLUT4 to the plasma membrane requires phosphatidylinositol (PI) 3-kinase activation-mediated generation of phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and subsequent activation of Akt. Previous studies suggested that skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) has negative effects on the regulation of insulin signalling in the skeletal muscle cells. Here, we compared its effects on insulin signalling by selective inhibition of SKIP, SHIP2 and PTEN by short interfering RNA in the C2C12 myoblast cells. Suppression of SKIP significantly increased the insulin-stimulated PIP(3) levels and Akt phosphorylation. Furthermore, silencing of SKIP, but not of PTEN, increased the insulin-dependent recruitment of GLUT4 vesicles to the plasma membrane. Taken together, these results imply that SKIP negatively regulates insulin signalling and glucose uptake by inhibiting GLUT4 docking and/or fusion to the plasma membrane.

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High-throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers.

Publication Date: 2012 Jan 14 PMID: 22247556
Authors: Faries, K. M. - Kressel, L. L. - Wander, M. J. - Holten, D. - Laible, P. D. - Kirmaier, C. - Hanson, D. K.
Journal: J Biol Chem

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A-pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B-branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high-throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure/function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.

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Determinants of formin homology 1 (FH1) domain function in actin filament elongation by formins.

Publication Date: 2012 Jan 14 PMID: 22247555
Authors: Courtemanche, N. - Pollard, T. D.
Journal: J Biol Chem

Formin-mediated elongation of actin filaments proceeds via association of Formin Homology 2 (FH2) domain dimers with the barbed end of the filament, allowing subunit addition while remaining processively attached to the end. The flexible Formin Homology 1 (FH1) domain, located directly N-terminal to the FH2 domain, contains one or more stretches of polyproline that bind the actin-binding protein profilin. Diffusion of FH1 domains brings associated profilin-actin complexes into contact with the FH2-bound barbed end of the filament, thereby enabling direct transfer of actin. We investigated how the organization of the FH1 domain of budding yeast formin Bni1p determines the rates of profilin-actin transfer onto the end of the filament. Each FH1 domain transfers actin to the barbed end independently of the other and structural evidence suggests a preference for actin delivery from each FH1 domain to the closest long-pitch helix of the filament. The transfer reaction is diffusion-limited and influenced by the affinities of the FH1 polyproline tracks for profilin. Position-specific sequence variations optimize the efficiency of FH1-stimulated polymerization by binding profilin weakly near the FH2 domain and binding profilin more strongly farther away. FH1 domains of many other formins follow this organizational trend. This particular sequence architecture may optimize the efficiency of FH1-stimulated elongation.

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Characterizing Phospholamban to SERCA2a Binding Interactions in Human Cardiac Sarcoplasmic Reticulum Vesicles Using Chemical Cross-linking.

Publication Date: 2012 Jan 14 PMID: 22247554
Authors: Akin, B. L. - Jones, L. R.
Journal: J Biol Chem

Chemical cross-linking was used to study protein-binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys27 of PLB was cross-linked to the Ca2+ pump at the cytoplasmic extension of M4 (at or near Lys328) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 A). Cross-linking was augmented by ATP but abolished by Ca2+ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca2+ affinity conformation of the Ca2+-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a, and 2D12 stimulation of Ca2+-ATPase activity and Ca2+-transport. The results suggest that the inhibitory effect of PLB on Ca2+-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca2+ to the Ca2+ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca2+ pump are largely unchanged in failing myocardium.

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Fibronectin induces endothelial cell migration through beta1-integrin and Src dependent phosphorylation of fibroblast growth factor receptor-1 at tyrosines 653/654 and 766.

Publication Date: 2012 Jan 14 PMID: 22247553
Authors: Zou, L. - Cao, S. - Kang, N. - Huebert, R. C. - Shah, V. H.
Journal: J Biol Chem

The extracellular matrix microenvironment regulates cell phenotype and function. One mechanism by which this is achieved is the transactivation of receptor tyrosine kinases by specific matrix molecules. Here, we demonstrate that the provisional matrix protein, fibronectin (FN), activates fibroblast growth factor (FGF) receptor-1 (FGFR1) independent of FGF ligand in liver endothelial cells. FN activation of FGFR1 requires beta1-integrin as evidenced by neutralizing antibody and siRNA based studies. Complementary genetic and pharmacologic approaches identify that the non-receptor tyrosine kinase Src is required for FN transactivation of FGFR1. While FGF ligand induced phosphorylation of FGFR1 preferentially activates Erk, FN-induced phosphorylation of FGFR1 preferentially activates Akt, indicating differential downstream signaling of FGFR1 in response to alternate stimuli. Mutation analysis of known tyrosine residues of FGFR1 reveals that tyrosine 653/654 and 766 residues are required for FN-FGFR1 activation of Akt and chemotaxis. Thus, our study mechanistically dissects a new signaling pathway by which FN achieves endothelial cell chemotaxis, demonstrates how differential phosphorylation profiles of FGFR1 can achieve alternate downstream signals, and more broadly, highlights the diversity of mechanisms by which the extracellular matrix microenvironment regulates cell behavior through transactivation of receptor tyrosine kinases.

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