EMBO Journal

FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.

Publication Date: 2008 May 8 PMID: 18464795
Authors: Bechara, A. - Nawabi, H. - Moret, F. - Yaron, A. - Weaver, E. - Bozon, M. - Abouzid, K. - Guan, J. L. - Tessier-Lavigne, M. - Lemmon, V. - Castellani, V.
Journal: EMBO J

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A.

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3' adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria.

Publication Date: 2008 May 8 PMID: 18464794
Authors: Etheridge, R. D. - Aphasizheva, I. - Gershon, P. D. - Aphasizhev, R.
Journal: EMBO J

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post-transcriptionally and requires extensive U-insertion/deletion mRNA editing. In mitochondrial extracts, 3' adenylation reportedly influences degradation kinetics of synthetic edited and pre-edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A-tails typically found in mitochondrial mRNAs, and decreases the abundance of never-edited and edited transcripts. Pre-edited mRNAs are not destabilized by the lack of 3' adenylation, whereas short A-tails are required and sufficient to maintain the steady-state levels of partially edited, fully edited, and never-edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A-tail in edited molecules. Upon completion of the editing process, the short A-tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3' processing and editing of mitochondrial mRNAs in trypanosomes.

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Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes.

Publication Date: 2008 May 8 PMID: 18464793
Authors: Pisarev, A. V. - Kolupaeva, V. G. - Yusupov, M. M. - Hellen, C. U. - Pestova, T. V.
Journal: EMBO J

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

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NF-kappaB dictates the degradation pathway of IkappaBalpha.

Publication Date: 2008 May 7 PMID: 18461078
Authors: Mathes, E. - O'Dea, E. L. - Hoffmann, A. - Ghosh, G.
Journal: EMBO J

Correction to: The EMBO Journal, advance online publication 10 April 2008; doi:10.1038/emboj.2008.73.

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Down to atomic-scale intracellular water dynamics.

Publication Date: 2008 May 2 PMID: 18451876
Authors: Jasnin, M. - Moulin, M. - Haertlein, M. - Zaccai, G. - Tehei, M.
Journal: EMBO J

Water constitutes the intracellular matrix in which biological molecules interact. Understanding its dynamic state is a main scientific challenge, which continues to provoke controversy after more than 50 years of study. We measured water dynamics in vivo in the cytoplasm of Escherichia coli by using neutron scattering and isotope labelling. Experimental timescales covered motions from pure water to interfacial water, on an atomic length scale. In contrast to the widespread opinion that water is 'tamed' by macromolecular confinement, the measurements established that water diffusion within the bacteria is similar to that of pure water at physiological temperature.

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Biological function in a non-native partially folded state of a protein.

Publication Date: 2008 May 1 PMID: 18451804
Authors: Bemporad, F. - Gsponer, J. - Hopearuoho, H. I. - Plakoutsi, G. - Stati, G. - Stefani, M. - Taddei, N. - Vendruscolo, M. - Chiti, F.
Journal: EMBO J

As structural flexibility is known to be required for enzyme catalysis and pattern recognition and a significant fraction of eukaryotic proteins appear to be unfolded or contain unstructured regions, biological activity of conformational states distinct from fully folded structures could be more common than previously thought. By applying a procedure that allows the recovery of enzymatic activity to be monitored in real time, we show that a non-native state populated transiently during folding of the acylphosphatase from Sulfolobus solfataricus is enzymatically active. The structural characterization of this partially folded state reveals that enzymatic activity is possible even if the catalytic site is structurally heterogeneous, whereas the remainder of the structure acts as a scaffold. These results extend the spectrum of biological functions carried out in the absence of a folded state to include enzyme catalysis.

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A Wingless and Notch double-repression mechanism regulates G1-S transition in the Drosophila wing.

Publication Date: 2008 May 1 PMID: 18451803
Authors: Herranz, H. - Perez, L. - Martin, F. A. - Milan, M.
Journal: EMBO J

The control of tissue growth and patterning is orchestrated in various multicellular tissues by the coordinated activity of the signalling molecules Wnt/Wingless (Wg) and Notch, and mutations in these pathways can cause cancer. The role of these molecules in the control of cell proliferation and the crosstalk between their corresponding pathways remain poorly understood. Crosstalk between Notch and Wg has been proposed to organize pattern and growth in the Drosophila wing primordium. Here we report that Wg and Notch act in a surprisingly linear pathway to control G1-S progression. We present evidence that these molecules exert their function by regulating the expression of the dmyc proto-oncogene and the bantam micro-RNA, which positively modulated the activity of the E2F transcription factor. Our results demonstrate that Notch acts in this cellular context as a repressor of cell-cycle progression and Wg has a permissive role in alleviating Notch-mediated repression of G1-S progression in wing cells.

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Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation.

Publication Date: 2008 May 1 PMID: 18451802
Authors: Weber, A. - Marquardt, J. - Elzi, D. - Forster, N. - Starke, S. - Glaum, A. - Yamada, D. - Defossez, P. A. - Delrow, J. - Eisenman, R. N. - Christiansen, H. - Eilers, M.
Journal: EMBO J

In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and represses expression of P21CIP1 as part of a heterodimeric complex with Miz1. In vivo, expression of ZBTB4 is downregulated in advanced stages of multiple human tumours. In cell culture, depletion of ZBTB4 promotes cell cycle arrest in response to activation of p53 and suppresses apoptosis through regulation of P21CIP1, thereby promoting long-term cell survival. Our data suggest that Zbtb4 is a critical determinant of the cellular response to p53 activation and reinforce the notion that p21Cip1 can provide an essential survival signal in cells with activated p53.

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Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay.

Publication Date: 2008 May 1 PMID: 18451801
Authors: Kerenyi, Z. - Merai, Z. - Hiripi, L. - Benkovics, A. - Gyula, P. - Lacomme, C. - Barta, E. - Nagy, F. - Silhavy, D.
Journal: EMBO J

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

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Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres.

Publication Date: 2008 Apr 17 PMID: 18418389
Authors: Temime-Smaali, N. - Guittat, L. - Wenner, T. - Bayart, E. - Douarre, C. - Gomez, D. - Giraud-Panis, M. J. - Londono-Vallejo, A. - Gilson, E. - Amor-Gueret, M. - Riou, J. F.
Journal: EMBO J

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.

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Yeast and human Ysl2p/hMon2 interact with Gga adaptors and mediate their subcellular distribution.

Publication Date: 2008 Apr 17 PMID: 18418388
Authors: Singer-Kruger, B. - Lasic, M. - Burger, A. M. - Hausser, A. - Pipkorn, R. - Wang, Y.
Journal: EMBO J

The Gga proteins represent a family of ubiquitously expressed clathrin adaptors engaged in vesicle budding at the tubular endosomal network/trans Golgi network. Their membrane recruitment is commonly thought to involve interactions with Arf and signals in cargo through the so-called VHS domain. For yeast Gga proteins, however, partners binding to its VHS domain have remained elusive and Gga localization does not absolutely depend on Arf. Here, we demonstrate that yeast Gga recruitment relies on a network of interactions between the scaffold Ysl2p/Mon2p, the small GTPase Arl1p, and the flippase Neo1p. Deletion of either YSL2 or ARL1 causes mislocalization of Gga2p, whereas a neo1-69 mutant accumulates Gga2p on aberrant structures. Remarkably, Ysl2p directly interacts with human and yeast Ggas through the VHS domain, and binding to Gga proteins is also found for the human Ysl2p orthologue hMon2. Thus, Ysl2p represents an essential, evolutionarily conserved member of a network controlling direct binding and membrane docking of Ggas. Because activated Arl1p is part of the network that binds Gga2p, Arf and Arf-like GTPases may interact in a regulatory cascade.

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Hzf regulates adipogenesis through translational control of C/EBPalpha.

Publication Date: 2008 Apr 17 PMID: 18418387
Authors: Kawagishi, H. - Wakoh, T. - Uno, H. - Maruyama, M. - Moriya, A. - Morikawa, S. - Okano, H. - Sherr, C. J. - Takagi, M. - Sugimoto, M.
Journal: EMBO J

Adipocyte differentiation requires a well-defined programme of gene expression in which the transcription factor C/EBPalpha (CCAAT/enhancer-binding protein) has a central function. Here, we show that Hzf (haematopoietic zinc-finger), a previously identified p53 transcriptional target, regulates C/EBPalpha expression. Hzf is induced during differentiation of preadipocyte cell lines, and its suppression by short hairpin RNA disrupts adipogenesis. In Hzf's absence, expression of C/EBPalpha is severely impaired because of reduced translation of its mRNA. Hzf physically interacts with the 3' untranslated region of C/EBPalpha mRNA to enhance its translation. Taken together, these findings underscore a critical role of Hzf in the adipogenesis regulatory cascade.

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Essential role of the chaperonin folding compartment in vivo.

Publication Date: 2008 Apr 17 PMID: 18418386
Authors: Tang, Y. C. - Chang, H. C. - Chakraborty, K. - Hartl, F. U. - Hayer-Hartl, M.
Journal: EMBO J

The GroEL/GroES chaperonin system of Escherichia coli forms a nano-cage allowing single protein molecules to fold in isolation. However, as the chaperonin can also mediate folding independently of substrate encapsulation, it remained unclear whether the folding cage is essential in vivo. To address this question, we replaced wild-type GroEL with mutants of GroEL having either a reduced cage volume or altered charge properties of the cage wall. A stepwise reduction in cage size resulted in a gradual loss of cell viability, although the mutants bound non-native protein efficiently. Strikingly, a mild reduction in cage size increased the yield and the apparent rate of green fluorescent protein folding, consistent with the view that an effect of steric confinement can accelerate folding. As shown in vitro, the observed acceleration of folding was dependent on protein encapsulation by GroES but independent of GroES cycling regulated by the GroEL ATPase. Altering the net-negative charge of the GroEL cage wall also strongly affected chaperonin function. Based on these findings, the GroEL/GroES compartment is essential for protein folding in vivo.

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Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3.

Publication Date: 2008 Apr 17 PMID: 18418385
Authors: Meyer, K. D. - Donner, A. J. - Knuesel, M. T. - York, A. G. - Espinosa, J. M. - Taatjes, A. D.
Journal: EMBO J

The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative 'T/G-Mediator' complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G-Mediator, cdk8 phosphorylates serine-10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co-occupancy of T/G-Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G-Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8-Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G-Mediator directs early events-such as modification of chromatin templates-in transcriptional activation.

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Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria.

Publication Date: 2008 Apr 17 PMID: 18418384
Authors: Popov-Celeketic, D. S. - Mapa, K. - Neupert, W. - Mokranjac, D.
Journal: EMBO J

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.

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Novel TCF-binding sites specify transcriptional repression by Wnt signalling.

Publication Date: 2008 Apr 17 PMID: 18418383
Authors: Blauwkamp, T. A. - Chang, M. V. - Cadigan, K. M.
Journal: EMBO J

Both transcriptional activation and repression have essential functions in maintaining proper spatial and temporal control of gene expression. Although Wnt signalling is often associated with gene activation, we have identified several directly repressed targets of Wnt signalling in Drosophila. Here, we explore how individual Wnt target genes are specified for signal-induced activation or repression. Similar to activation, repression required binding of Armadillo (Arm) to the N terminus of TCF. However, TCF/Arm mediated repression by binding to DNA motifs that are markedly different from typical TCF-binding sites. Conversion of the novel motifs to standard TCF-binding sites reversed the mode of regulation, resulting in Wnt-mediated activation instead of repression. A mutant form of Arm defective in activation was still functional for repression, indicating that distinct domains of the protein are required for each activity. This study suggests that the sequence of TCF-binding sites allosterically regulates the TCF/Arm complex to effect either transcriptional activation or repression.

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Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres.

Publication Date: 2008 Apr 17 PMID: 18418382
Authors: Lazzaro, F. - Sapountzi, V. - Granata, M. - Pellicioli, A. - Vaze, M. - Haber, J. E. - Plevani, P. - Lydall, D. - Muzi-Falconi, M.
Journal: EMBO J

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.

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Oxygen-regulated degradation of fission yeast SREBP by Ofd1, a prolyl hydroxylase family member.

Publication Date: 2008 Apr 17 PMID: 18418381
Authors: Hughes, B. T. - Espenshade, P. J.
Journal: EMBO J

Sre1, the fission yeast sterol regulatory element binding protein, is an endoplasmic reticulum membrane-bound transcription factor that responds to changes in oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. Under low oxygen, Sre1 is proteolytically cleaved and the released N-terminal transcription factor (Sre1N) activates gene expression essential for hypoxic growth. Here, we describe an oxygen-dependent mechanism for regulation of Sre1 that is independent of sterol-regulated proteolysis. Using yeast expressing only Sre1N, we show that Sre1N turnover is regulated by oxygen. Ofd1, an uncharacterized prolyl 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase, accelerates Sre1N degradation in the presence of oxygen. However, unlike the prolyl 4-hydroxylases that regulate mammalian hypoxia-inducible factor, Ofd1 uses multiple domains to regulate Sre1N degradation by oxygen; the Ofd1 N-terminal dioxygenase domain is required for oxygen sensing and the Ofd1 C-terminal domain accelerates Sre1N degradation. Our data support a model whereby the Ofd1 N-terminal dioxygenase domain is an oxygen sensor that regulates the activity of the C-terminal degradation domain.

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A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice.

Publication Date: 2008 May 7 PMID: 18401344
Authors: Phillips, J. M. - Murray, P. S. - Murray, D. - Vogt, V. M.
Journal: EMBO J

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.

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Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells.

Publication Date: 2008 May 7 PMID: 18401343
Authors: Waghmare, S. K. - Bansal, R. - Lee, J. - Zhang, Y. V. - McDermitt, D. J. - Tumbar, T.
Journal: EMBO J

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

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NF-kappaB dictates the degradation pathway of IkappaBalpha.

Publication Date: 2008 May 7 PMID: 18401342
Authors: Mathes, E. - O'Dea, E. L. - Hoffmann, A. - Ghosh, G.
Journal: EMBO J

IkappaB proteins are known as the regulators of NF-kappaB activity. They bind tightly to NF-kappaB dimers, until stimulus-responsive N-terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IkappaBalpha is an unstable protein whose rapid degradation is slowed upon binding to NF-kappaB, but it is not known what dynamic mechanisms control the steady-state level of total IkappaBalpha. Here, we show clearly that two degradation pathways control the level of IkappaBalpha. Free IkappaBalpha degradation is not controlled by IKK or ubiquitination but intrinsically, by the C-terminal sequence known as the PEST domain. NF-kappaB binding to IkappaBalpha masks the PEST domain from proteasomal recognition, precluding ubiquitin-independent degradation; bound IkappaBalpha then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IkappaBalpha protein; alteration of free IkappaBalpha degradation dampens NF-kappaB activation. In addition, we find that both free and bound IkappaBalpha are similar substrates for IKK, and the preferential phosphorylation of NF-kappaB-bound IkappaBalpha is due to stabilization of IkappaBalpha by NF-kappaB.

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The FoxO3a gene is a key negative target of canonical Notch signalling in the keratinocyte UVB response.

Publication Date: 2008 Apr 23 PMID: 18388864
Authors: Mandinova, A. - Lefort, K. - Tommasi di Vignano, A. - Stonely, W. - Ostano, P. - Chiorino, G. - Iwaki, H. - Nakanishi, J. - Dotto, G. P.
Journal: EMBO J

Notch signalling has an important role in skin homeostasis, promoting keratinocyte differentiation and suppressing tumorigenesis. Here we show that this pathway also has an essential anti-apoptotic function in the keratinocyte UVB response. Notch1 expression and activity are significantly induced, in a p53-dependent manner, by UVB exposure of primary keratinocytes as well as intact epidermis of both mouse and human origin. The apoptotic response to UVB is increased by deletion of the Notch1 gene or down-modulation of Notch signalling by pharmacological inhibition or genetic suppression of 'canonical' Notch/CSL/MAML1-dependent transcription. Conversely, Notch activation protects keratinocytes against apoptosis through a mechanism that is not linked to Notch-induced cell cycle withdrawal or NF-kappaB activation. Rather, transcription of FoxO3a, a key pro-apoptotic gene, is under direct negative control of Notch/HERP transcription in keratinocytes, and upregulation of this gene accounts for the increased susceptibility to UVB of cells with suppressed Notch signalling. Thus, the canonical Notch/HERP pathway functions as a protective anti-apoptotic mechanism in keratinocytes through negative control of FoxO3a expression.

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A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor.

Publication Date: 2008 May 7 PMID: 18388863
Authors: Turner, N. C. - Lord, C. J. - Iorns, E. - Brough, R. - Swift, S. - Elliott, R. - Rayter, S. - Tutt, A. N. - Ashworth, A.
Journal: EMBO J

Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.

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Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning.

Publication Date: 2008 May 7 PMID: 18388862
Authors: Rondard, P. - Huang, S. - Monnier, C. - Tu, H. - Blanchard, B. - Oueslati, N. - Malhaire, F. - Li, Y. - Trinquet, E. - Labesse, G. - Pin, J. P. - Liu, J.
Journal: EMBO J

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.

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Mus81 is essential for sister chromatid recombination at broken replication forks.

Publication Date: 2008 May 7 PMID: 18388861
Authors: Roseaulin, L. - Yamada, Y. - Tsutsui, Y. - Russell, P. - Iwasaki, H. - Arcangioli, B.
Journal: EMBO J

Recombination is essential for the recovery of stalled/collapsed replication forks and therefore for the maintenance of genomic stability. The situation becomes critical when the replication fork collides with an unrepaired single-strand break and converts it into a one-ended double-strand break. We show in fission yeast that a unique broken replication fork requires the homologous recombination (HR) enzymes for cell viability. Two structure-specific heterodimeric endonucleases participate in two different resolution pathways. Mus81/Eme1 is essential when the sister chromatid is used for repair; conversely, Swi9/Swi10 is essential when an ectopic sequence is used for repair. Consequently, the utilization of these two HR modes of resolution mainly relies on the ratio of unique and repeated sequences present in various eukaryotic genomes. We also provide molecular evidence for sister recombination intermediates. These findings demonstrate that Mus81/Eme1 is the dedicated endonuclease that resolves sister chromatid recombination intermediates during the repair of broken replication forks.

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Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria.

Publication Date: 2008 May 7 PMID: 18388860
Authors: Mullineaux, C. W. - Mariscal, V. - Nenninger, A. - Khanum, H. - Herrero, A. - Flores, E. - Adams, D. G.
Journal: EMBO J

Heterocyst-forming filamentous cyanobacteria are true multicellular prokaryotes, in which heterocysts and vegetative cells have complementary metabolism and are mutually dependent. The mechanism for metabolite exchange between cells has remained unclear. To gain insight into the mechanism and kinetics of metabolite exchange, we introduced calcein, a 623-Da fluorophore, into the Anabaena cytoplasm. We used fluorescence recovery after photobleaching to quantify rapid diffusion of this molecule between the cytoplasms of all the cells in the filament. This indicates nonspecific intercellular channels allowing the movement of molecules from cytoplasm to cytoplasm. We quantify rates of molecular exchange as filaments adapt to diazotrophic growth. Exchange among vegetative cells becomes faster as filaments differentiate, becoming considerably faster than exchange with heterocysts. Slower exchange is probably a price paid to maintain a microaerobic environment in the heterocyst. We show that the slower exchange is partly due to the presence of cyanophycin polar nodules in heterocysts. The phenotype of a null mutant identifies FraG (SepJ), a membrane protein localised at the cell-cell interface, as a strong candidate for the channel-forming protein.

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Akt- and Foxo1-interacting WD-repeat-FYVE protein promotes adipogenesis.

Publication Date: 2008 May 7 PMID: 18388859
Authors: Fritzius, T. - Moelling, K.
Journal: EMBO J

We have previously identified a protein, consisting of seven WD-repeats, forming a putative beta-propeller, and an FYVE domain, ProF, which is highly expressed in 3T3-L1 cells, a cell line that can be differentiated into adipocytes. We recently found ProF to interact with the kinases Akt and protein kinase Czeta. Here we demonstrate that ProF is a positive regulator of adipogenesis. Knockdown of ProF by RNA interference leads to decreased adipocyte differentiation. This is shown by reduced lipid accumulation, decreased expression of the differentiation markers PPARgamma and C/EBPalpha, and reduced glucose uptake in differentiated cells. Furthermore, ProF overexpression leads to increased adipogenesis. ProF binds to the transcription factor Foxo1 (Forkhead box O1), a negative regulator of insulin action and adipogenesis, and facilitates the phosphorylation and thus inactivation of Foxo1 by Akt. Additionally, dominant-negative Foxo1 restores adipogenesis in ProF knockdown cells. Thus, ProF modulates Foxo1 phosphorylation by Akt, promoting adipocyte differentiation. Furthermore, ProF might be involved in metabolic disorders such as diabetes.

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Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response.

Publication Date: 2008 Apr 23 PMID: 18388858
Authors: Jang, S. - Marchal, V. - Panigrahi, K. C. - Wenkel, S. - Soppe, W. - Deng, X. W. - Valverde, F. - Coupland, G.
Journal: EMBO J

The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

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Motor step size and ATP coupling efficiency of the dsDNA translocase EcoR124I.

Publication Date: 2008 May 7 PMID: 18388857
Authors: Seidel, R. - Bloom, J. G. - Dekker, C. - Szczelkun, M. D.
Journal: EMBO J

The Type I restriction-modification enzyme EcoR124I is an archetypical helicase-based dsDNA translocase that moves unidirectionally along the 3'-5' strand of intact duplex DNA. Using a combination of ensemble and single-molecule measurements, we provide estimates of two physicochemical constants that are fundamental to a full description of motor protein activity-the ATP coupling efficiency (the number of ATP consumed per base pair) and the step size (the number of base pairs transported per motor step). Our data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumed per base pair slightly exceeds unity. Our observations form a framework for understanding energy coupling in a great many other motors that translocate along dsDNA rather than ssDNA.

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Activation of TRPP2 through mDia1-dependent voltage gating.

Publication Date: 2008 May 7 PMID: 18388856
Authors: Bai, C. X. - Kim, S. - Li, W. P. - Streets, A. J. - Ong, A. C. - Tsiokas, L.
Journal: EMBO J

The TRPP2 cation channel is directly responsible for approximately 15% of all cases of autosomal dominant polycystic kidney disease. However, the mechanisms underlying fundamental properties of TRPP2 regulation, such as channel gating and activation, are unknown. We have shown that TRPP2 was activated by EGF and physically interacted with the mammalian diaphanous-related formin 1 (mDia1), a downstream effector of RhoA. Now, we show that mDia1 regulates TRPP2 by specifically blocking its activity at negative but not positive potentials. The voltage-dependent unblock of TRPP2 by mDia1 at positive potentials is mediated through RhoA-induced molecular switching of mDia1 from its autoinhibited state at negative potentials to its activated state at positive potentials. Under physiological resting potentials, EGF activates TRPP2 by releasing the mDia1-dependent block through the activation of RhoA. Our data reveal a new role of mDia1 in the regulation of ion channels and suggest a molecular basis for the voltage-dependent gating of TRP channels.

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G protein-coupled receptor kinase 2 positively regulates epithelial cell migration.

Publication Date: 2008 Apr 23 PMID: 18369319
Authors: Penela, P. - Ribas, C. - Aymerich, I. - Eijkelkamp, N. - Barreiro, O. - Heijnen, C. J. - Kavelaars, A. - Sanchez-Madrid, F. - Mayor, F. Jr
Journal: EMBO J

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.

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A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype.

Publication Date: 2008 Apr 23 PMID: 18369318
Authors: Kim, H. J. - Li, Q. - Tjon-Kon-Sang, S. - So, I. - Kiselyov, K. - Soyombo, A. A. - Muallem, S.
Journal: EMBO J

TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca(2+)-permeable channel with a unique form of regulation by extracytosolic (luminal) H(+) (H(+)(e-cyto)). Regulation by H(+)(e-cyto) is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H(+)(e-cyto) to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H(+)(e-cyto), and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H(+)(e-cyto) are altered by both the alpha-helix-destabilizing A419G and the alpha-helix-favouring A419M and A419K. These findings suggest that regulation of TRPML3 by H(+)(e-cyto) is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication.

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Drosophila EGFR signalling is modulated by differential compartmentalization of Rhomboid intramembrane proteases.

Publication Date: 2008 Apr 23 PMID: 18369317
Authors: Yogev, S. - Schejter, E. D. - Shilo, B. Z.
Journal: EMBO J

We explore the role of differential compartmentalization of Rhomboid (Rho) proteases that process the Drosophila EGF receptor ligands, in modulating the amount of secreted ligand and consequently the level of EGF receptor (EGFR) activation. The mSpitz ligand precursor is retained in the ER, and is trafficked by the chaperone Star to a late compartment of the secretory pathway, where Rho-1 resides. This work demonstrates that two other Rho proteins, Rho-2 and Rho-3, which are expressed in the germ line and in the developing eye, respectively, cleave the Spitz precursor and Star already in the ER, in addition to their activity in the late compartment. This property attenuates EGFR activation, primarily by compromising the amount of chaperone that can productively traffic the ligand precursor to the late compartment, where cleavage and subsequent secretion take place. These observations identify changes in intracellular compartment localization of Rho proteins as a basis for signal attenuation, in tissues where EGFR activation must be highly restricted in space and time.

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Peptides released from reovirus outer capsid form membrane pores that recruit virus particles.

Publication Date: 2008 Apr 23 PMID: 18369316
Authors: Ivanovic, T. - Agosto, M. A. - Zhang, L. - Chandran, K. - Harrison, S. C. - Nibert, M. L.
Journal: EMBO J

Nonenveloped animal viruses must disrupt or perforate a cell membrane during entry. Recent work with reovirus has shown formation of size-selective pores in RBC membranes in concert with structural changes in capsid protein mu1. Here, we demonstrate that mu1 fragments released from reovirus particles are sufficient for pore formation. Both myristoylated N-terminal fragment mu1N and C-terminal fragment phi are released from particles. Both also associate with RBC membranes and contribute to pore formation in the absence of particles, but mu1N has the primary and sufficient role. Particles with a mutant form of mu1, unable to release mu1N or form pores, lack the ability to associate with membranes. They are, however, recruited by pores preformed with peptides released from wild-type particles or with synthetic mu1N. The results provide evidence that docking to membrane pores by virus particles may be a next step in membrane penetration after pore formation by released peptides.

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The kinase Syk as an adaptor controlling sustained calcium signalling and B-cell development.

Publication Date: 2008 May 7 PMID: 18369315
Authors: Kulathu, Y. - Hobeika, E. - Turchinovich, G. - Reth, M.
Journal: EMBO J

Upon B-cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (SLP-65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP-65 is not only a downstream target and substrate of Syk but also a direct binding-partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP-65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP-65 complex are defective in BCR-induced extracellular signal-regulated kinase, nuclear factor kappa B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B-cell development. Furthermore, we show that formation of the Syk/SLP-65 complex is required for sustained Ca(2+) responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane-bound Syk/SLP-65 complex controlling sustained signalling and calcium influx.

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A crucial role of WW45 in developing epithelial tissues in the mouse.

Publication Date: 2008 Apr 23 PMID: 18369314
Authors: Lee, J. H. - Kim, T. S. - Yang, T. H. - Koo, B. K. - Oh, S. P. - Lee, K. P. - Oh, H. J. - Lee, S. H. - Kong, Y. Y. - Kim, J. M. - Lim, D. S.
Journal: EMBO J

The role and molecular mechanisms of a new Hippo signalling pathway are not fully understood in mammals. Here, we generated mice that lack WW45 and revealed a crucial role for WW45 in cell-cycle exit and epithelial terminal differentiation. Many organs in the mutant mouse embryos displayed hyperplasia accompanied by defects in terminal differentiation of epithelial progenitor cells owing to impaired proliferation arrest rather than intrinsic acceleration of proliferation during differentiation. Importantly, the MST1 signalling pathway is specifically activated in differentiating epithelial cells. Moreover, WW45 is required for MST1 activation and translocation to the nucleus for subsequent LATS1/2 activation upon differentiation signal. LATS1/2 phosphorylates YAP, which, in turn, translocates from the nucleus into the cytoplasm, resulting in cell-cycle exit and terminal differentiation of epithelial progenitor cells. Collectively, these data provide compelling evidence that WW45 is a key mediator of MST1 signalling in the coordinate coupling of proliferation arrest with terminal differentiation for proper epithelial tissue development in mammals.

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The initiation factor eIF3-f is a major target for Atrogin1/MAFbx function in skeletal muscle atrophy.

Publication Date: 2008 Apr 23 PMID: 18354498
Authors: Lagirand-Cantaloube, J. - Offner, N. - Csibi, A. - Leibovitch, M. P. - Batonnet-Pichon, S. - Tintignac, L. A. - Segura, C. T. - Leibovitch, S. A.
Journal: EMBO J

In response to cancer, AIDS, sepsis and other systemic diseases inducing muscle atrophy, the E3 ubiquitin ligase Atrogin1/MAFbx (MAFbx) is dramatically upregulated and this response is necessary for rapid atrophy. However, the precise function of MAFbx in muscle wasting has been questioned. Here, we present evidence that during muscle atrophy MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3-f) for ubiquitination and degradation by the proteasome. Ectopic expression of MAFbx in myotubes induces atrophy and degradation of eIF3-f. Conversely, blockade of MAFbx expression by small hairpin RNA interference prevents eIF3-f degradation in myotubes undergoing atrophy. Furthermore, genetic activation of eIF3-f is sufficient to cause hypertrophy and to block atrophy in myotubes, whereas genetic blockade of eIF3-f expression induces atrophy in myotubes. Finally, eIF3-f induces increasing expression of muscle structural proteins and hypertrophy in both myotubes and mouse skeletal muscle. We conclude that eIF3-f is a key target that accounts for MAFbx function during muscle atrophy and has a major role in skeletal muscle hypertrophy. Thus, eIF3-f seems to be an attractive therapeutic target.

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Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein.

Publication Date: 2008 Apr 23 PMID: 18354496
Authors: Sato, M. - Sato, K. - Liou, W. - Pant, S. - Harada, A. - Grant, B. D.
Journal: EMBO J

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.

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Epigenetic regulation of TTF-I-mediated promoter-terminator interactions of rRNA genes.

Publication Date: 2008 Apr 23 PMID: 18354495
Authors: Nemeth, A. - Guibert, S. - Tiwari, V. K. - Ohlsson, R. - Langst, G.
Journal: EMBO J

Ribosomal RNA synthesis is the eukaryotic cell's main transcriptional activity, but little is known about the chromatin domain organization and epigenetics of actively transcribed rRNA genes. Here, we show epigenetic and spatial organization of mouse rRNA genes at the molecular level. TTF-I-binding sites subdivide the rRNA transcription unit into functional chromatin domains and sharply delimit transcription factor occupancy. H2A.Z-containing nucleosomes occupy the spacer promoter next to a newly characterized TTF-I-binding site. The spacer and the promoter proximal TTF-I-binding sites demarcate the enhancer. DNA from both the enhancer and the coding region is hypomethylated in actively transcribed repeats. 3C analysis revealed an interaction between promoter and terminator regions, which brings the beginning and end of active rRNA genes into close contact. Reporter assays show that TTF-I mediates this interaction, thereby linking topology and epigenetic regulation of the rRNA genes.

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