Biochimica et Biophysica Acta

Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants.

Publication Date: 2008 Apr 25 PMID: 18477484
Authors: Nakamura, T. - Sakai, M. - Sadakane, Y. - Haga, T. - Goto, Y. - Kinouchi, T. - Saito, T. - Fujii, N.
Journal: Biochim Biophys Acta

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.

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Mitochondrial death pathways in yeast and mammalian cells.

Publication Date: 2008 May 2 PMID: 18477482
Authors: Cheng, W. C. - Leach, K. M. - Hardwick, J. M.
Journal: Biochim Biophys Acta

In mammals, mitochondria are important mediators of programmed cell death, and this process is often regulated by Bcl-2 family proteins. However, a role for mitochondria-mediated cell death in non-mammalian species is more controversial. New evidence from a variety of sources suggests that mammalian mitochondrial fission/division proteins also have the capacity to promote programmed cell death, which may involve interactions with Bcl-2 family proteins. Homologues of these fission factors and several additional mammalian cell death regulators are conserved in flies, worms and yeast, and have been suggested to regulate programmed cell death in these species as well. However, the molecular mechanisms by which these phylogenetically conserved proteins contribute to cell death are not known for any species. Some have taken the conserved pro-death activity of mitochondrial fission factors to mean that mitochondrial fission per se, or failed attempts to undergo fission, are directly involved in cell death. Other evidence suggests that the fission function and the cell death function of these factors are separable. Here we consider the evidence for these arguments and their implications regarding the origins of programmed cell death.

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The V108M mutation decreases the structural stability of catechol O-methyltransferase.

Publication Date: 2008 Apr 24 PMID: 18474266
Authors: Rutherford, K. - Alphandery, E. - McMillan, A. - Daggett, V. - Parson, W. W.
Journal: Biochim Biophys Acta

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and in their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

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Guanylyl cyclase and protein kinase G mediate nitric oxide suppression of 5-lipoxygenase metabolism in rat alveolar macrophages.

Publication Date: 2008 Apr 24 PMID: 18474265
Authors: Coffey, M. J. - Phare, S. M. - Luo, M. - Peters-Golden, M.
Journal: Biochim Biophys Acta

We have previously demonstrated that exogenous nitric oxide (NO) directly inhibits alveolar macrophage (AM) cell-free activity of the enzyme 5-lipoxygenase (5-LO), thereby inhibiting metabolism of arachidonic acid to the important proinflammatory lipid mediators, leukotrienes (LT). Here, we explored the possibility that NO indirectly inhibited AM LT synthesis via activation of soluble guanylyl cyclase (sGC) in rat AM. The selective sGC inhibitor, LY83583, abrogated the suppression of cellular LT synthesis elicited by either exogenous or endogenous NO. A non-NO-dependent activator of sGC, YC-1, also inhibited macrophage LT synthesis. We next determined if sGC-mediated suppression of AM LT synthesis was dependent on protein kinase G (cGK). The selective cGK inhibitor, KT5823, reversed the suppression of cellular 5-LO metabolism following treatment with exogenous NO and YC-1. cGK1 activation resulted in phosphorylation of 5-LO. In contrast to peritoneal macrophages, AM exhibited localization of sGC, cGK1 and cGKII to the cell nucleus. In summary, in addition to its direct effects, NO-induced suppression of 5-LO action can be mediated indirectly through activation of the sGC and cGK pathways in AM. The nuclear localization of enzymes sGC, CGK1 and cGKII in the AM, which also demonstrates preferential nuclear 5-LO expression, may confer tighter regulation of LT synthesis.

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Human lens lipids differ markedly from those of commonly used experimental animals.

Publication Date: 2008 Apr 16 PMID: 18474264
Authors: Deeley, J. M. - Mitchell, T. W. - Wei, X. - Korth, J. - Nealon, J. R. - Blanksby, S. J. - Truscott, R. J.
Journal: Biochim Biophys Acta

Electrospray ionisation tandem mass spectrometry has allowed the unambiguous identification and quantification of individual lens phospholipids in human and six animal models. Using this approach ca. 100 unique phospholipids have been characterised. Parallel analysis of the same lens extracts by a novel direct-insertion electron-ionization technique found the cholesterol content of human lenses to be significantly higher (ca. 6 times) than lenses from the other animals. The most abundant phospholipids in all the lenses examined were choline-containing phospholipids. In rat, mouse, sheep, cow, pig and chicken, these were present largely as phosphatidylcholines, in contrast 66% of the total phospholipid in Homo sapiens was sphingomyelin, with the most abundant being dihydrosphingomyelins, in particular SM(d18:0/16:0) and SM(d18:0/24:1). The abundant glycerophospholipids within human lenses were found to be predominantly phosphatidylethanolamines and phosphatidylserines with surprisingly high concentrations of ether-linked alkyl chains identified in both classes. This study is the first to identify the phospholipid class (head-group) and assign the constituent fatty acid(s) for each lipid molecule and to quantify individual lens phospholipids using internal standards. These data clearly indicate marked differences in the membrane lipid composition of the human lens compared to commonly used animal models and thus predict a significant variation in the membrane properties of human lens fibre cells compared to those of other animals.

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The regulation of sister chromatid cohesion.

Publication Date: 2008 Apr 24 PMID: 18474253
Authors: Losada, A.
Journal: Biochim Biophys Acta

Sister chromatid cohesion is a major feature of the eukaryotic chromosome. It entails the formation of a physical linkage between the two copies of a chromosome that result from the duplication process. This linkage must be maintained until chromosome segregation takes place in order to ensure the accurate distribution of the genomic information. Cohesin, a multiprotein complex conserved from yeast to humans, is largely responsible for sister chromatid cohesion. Other cohesion factors regulate the interaction of cohesin with chromatin as well as the establishment and dissolution of cohesion. In addition, the presence of cohesin throughout the genome appears to influence processes other than chromosome segregation, such as transcription and DNA repair. In this review I summarize recent advances in our understanding of cohesin function and regulation in mitosis, and discuss the consequences of impairing the cohesion process at the level of the whole organism.

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Crosstalk between Nap1 protein and Cds1 checkpoint kinase to maintain chromatin integrity.

Publication Date: 2008 Apr 16 PMID: 18474252
Authors: Grande, M. - Lambea, E. - Fajardo, A. - Lopez-Aviles, S. - Kellogg, D. - Aligue, R.
Journal: Biochim Biophys Acta

The nucleosome assembly protein Nap1 has been implicated in various cellular functions such as histone shuttling into the nucleus, nucleosome assembly, chromatin remodelling, transcriptional control and cell-cycle regulation in Saccharomyces cerevisiae. In Schizosaccharomyces pombe nap1 null mutant cells are viable but they showed a delay in the onset of mitosis which is rescued by the absence of the replication Cds1 checkpoint kinase. In contrast the absence of the DNA-damage Chk1 checkpoint kinase is unable to rescue the delay. Moreover, the double nap1 cds1 mutant cells lose viability and cells show positive H2AX phosphorylation, suggesting that the viability of nap1-deleted cells is due to the Cds1 kinase. We also show that overexpression of Nap1 protein blocks the cell cycle in the G1 phase.

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The Rieske/cytochrome b complex of Heliobacteria.

Publication Date: 2008 Apr 23 PMID: 18474216
Authors: Ducluzeau, A. L. - Chenu, E. - Capowiez, L. - Baymann, F.
Journal: Biochim Biophys Acta

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b(6,) a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b(6)f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c(i) is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b(6)f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.

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The 2-methoxy group of ubiquinone is essential for function of the acceptor quinones in reaction centers from Rba. sphaeroides.

Publication Date: 2008 Apr 23 PMID: 18474215
Authors: Wraight, C. A. - Vakkasoglu, A. - Poluektov, Y. - Mattis, A. - Barbara, S. - Lipshutz, B.
Journal: Biochim Biophys Acta

The orientation of a methoxy substituent is known to substantially influence the electron affinity and vibrational spectroscopy of benzoquinones, and has been suggested to be important in determining the function of ubiquinone as a redox cofactor in bioenergetics. Ubiquinone functions as both the primary (Q(A)) and secondary (Q(B)) quinone in the reaction centers of many purple photosynthetic bacteria, and is almost unique in its ability to establish the necessary redox free energy gap for 1-electron transfer between them. The role of the methoxy substitution in this requirement was examined using monomethoxy analogues of ubiquinone-4 - 2-methoxy-3,5-dimethyl-6-isoprenyl-1,4-benzoquinone (2-MeO-Q) and 3-methoxy-2,5-dimethyl-6-isoprenyl-1,4-benzoquinone (3-MeO-Q). Only 2-MeO-Q was able to simultaneously act as Q(A) and Q(B) and the necessary redox potential tuning was shown to occur in the Q(B) site. In the absence of active Q(B), the IR spectrum of the monomethoxy quinones was examined in vitro and in the Q(A) site, and a novel distinction between the two methoxy groups was tentatively identify, consistent with the unique role of the 2-methoxy group in distinguishing Q(A) and Q(B) functionality.

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Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase.

Publication Date: 2008 Apr 22 PMID: 18472025
Authors: Ohmae, E. - Tatsuta, M. - Abe, F. - Kato, C. - Tanaka, N. - Kunugi, S. - Gekko, K.
Journal: Biochim Biophys Acta

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.

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Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and alpha-B-crystallin.

Publication Date: 2008 Apr 18 PMID: 18472024
Authors: Agnetti, G. - Bezstarosti, K. - Dekkers, D. H. - Verhoeven, A. J. - Giordano, E. - Guarnieri, C. - Caldarera, C. M. - Van Eyk, J. - Lamers, J. M.
Journal: Biochim Biophys Acta

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as alpha-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and alpha-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.

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RPA nucleic acid-binding properties of IFI16-HIN200.

Publication Date: 2008 Apr 22 PMID: 18472023
Authors: Yan, H. - Dalal, K. - Hon, B. K. - Youkharibache, P. - Pio, F.
Journal: Biochim Biophys Acta

InterFeron-gamma Inducible protein 16 (IFI16) belongs to the interferon inducible HIN200 protein family that contains transcriptional regulators linked to cell cycle regulation and differentiation. All family members contain at most two domains of 200 amino acids called HIN200 each containing two Oligonucleotide/Oligosaccharide Binding (OB) folds. IFI16 is involved in transcriptional repression and is a component of the DNA repair multi-protein complex known as BASC, which forms after UV-induced DNA damage. In this study, we used fold recognition and biophysical approaches as a tool to infer and validate function to the HIN200 domain. Since the best template to model IFI16-HIN200 is with Replication Protein A (RPA) in complex with single-stranded nucleic acids we tested six RPA nucleic acid-binding characteristics for IFI16-HIN200. Our results indicate that IFI16-HIN200 is an RPA-like, OB-fold, nucleic acid-binding protein that binds to ssDNA with higher affinity than to dsDNA, recognizes ssDNA in the same orientation as RPA, oligomerizes upon ssDNA binding, wraps and stretches ssDNA, but does not destabilize dsDNA. We finally propose a framework model explaining how the HIN200 domain could prevent ssDNA from re-annealing.

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Plasticity-related genes (PRGs/LRPs): A brain-specific class of lysophospholipid-modifying proteins.

Publication Date: 2008 Apr 22 PMID: 18472022
Authors: Brauer, A. U. - Nitsch, R.
Journal: Biochim Biophys Acta

Recently, a set of five brain-specifically expressed membrane proteins, which define a novel subclass of the lipid phosphate phosphatases (LPP-)superfamily, has been identified, namely plasticity-related genes (PRGs/LRPs). The primary known significance of these genes is their involvement in regeneration processes and attenuation of effects induced by lysophosphatidic acid (LPA). LPA is key player in lysophospholipids, a hydrophilic group of lipids that have been recognized as important signaling molecules. It is a lipid mediator with a wide variety of biological actions, such as cell proliferation, migration and survival. Its extracellular effects are mediated through five distinct G-protein-coupled receptors (LPA(1-5)) and LPA therefore activates multiple signal transduction pathways. LPA signaling has been implicated in diverse processes, such as wound healing, brain development, vascular remodeling and tumor progression. LPA levels are controlled by enzymes that synthesize or degrade LPA and, thus, these enzymes also regulate many aspects of signaling transduction. Three LPPs and a splice variant have been demonstrated as deactivating LPA. Studies of PRGs indicate that this group of proteins may in fact serve as controllers of LPA and therefore opening the door to new therapeutic approaches.

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Sphingosine-1-phosphate signaling and biological activities in the cardiovascular system.

Publication Date: 2008 Apr 22 PMID: 18472021
Authors: Takuwa, Y. - Okamoto, Y. - Yoshioka, K. - Takuwa, N.
Journal: Biochim Biophys Acta

The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphingosine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P(1), S1P(2) and S1P(3). S1P(1) expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P(2), is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G(12/13)-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P(1) is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P(3) expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P(3) expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P(3), together with S1P(2) and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P(3) signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer.

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p53, cyclin-dependent kinase and abnormal amplification of centrosomes.

Publication Date: 2008 Apr 22 PMID: 18472015
Authors: Fukasawa, K.
Journal: Biochim Biophys Acta

Centrosomes play a critical role in formation of bipolar mitotic spindles, an essential event for accurate chromosome segregation into daughter cells. Numeral abnormalities of centrosomes (centrosome amplification) occur frequently in cancers, and are considered to be the major cause of chromosome instability, which accelerates acquisition of malignant phenotypes during tumor progression. Loss or mutational inactivation of p53 tumor suppressor protein, one of the most common mutations found in cancers, results in a high frequency of centrosome amplification in part via allowing the activation of the cyclin-dependent kinase (CDK) 2-cyclin E (as well as CDK2-cyclin A) which is a key factor for the initiation of centrosome duplication. In this review, the role of centrosome amplification in tumor progression, and mechanistic view of how centrosomes are amplified in cells through focusing on loss of p53 and aberrant activities of CDK2-cyclins will be discussed.

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Preventing aneuploidy: The contribution of mitotic checkpoint proteins.

Publication Date: 2008 Apr 22 PMID: 18472014
Authors: Suijkerbuijk, S. J. - Kops, G. J.
Journal: Biochim Biophys Acta

Aneuploidy, an abnormal number of chromosomes, is a trait shared by most solid tumors. Chromosomal instability (CIN) manifested as aneuploidy might promote tumorigenesis and cause increased resistance to anti-cancer therapies. The mitotic checkpoint or spindle assembly checkpoint is a major signaling pathway involved in the prevention of CIN. We review current knowledge on the contribution of misregulation of mitotic checkpoint proteins to tumor formation and will address to what extent this contribution is due to chromosome segregation errors directly. We propose that both checkpoint and non-checkpoint functions of these proteins contribute to the wide array of oncogenic phenotypes seen upon their misregulation.

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Interferon regulatory factor-1 (IRF-1) regulates VEGF-induced angiogenesis in HUVECs.

Publication Date: 2008 Apr 22 PMID: 18472010
Authors: Lee, J. H. - Chun, T. - Park, S. Y. - Rho, S. B.
Journal: Biochim Biophys Acta

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor and transcriptional modulator that can regulate gene expression involved in cell growth control, induction of apoptosis, and post-translation modification. In this study, we found that IRF-1 inhibits endothelial cell angiogenesis using human umbilical vein endothelial cell (HUVECs) culture system. In addition, IRF-1 directly inhibited the tube formation of endothelial cells on Matrigel and reduced the expression of p-Akt, and p-eNOS, which play a significant role in angiogenesis when stimulated by VEGF. We also demonstrate that C-terminal region including transactivation domain (TA) of IRF-1 functions as a signal for its angiostatic activity, and is spliced in human tumor tissues. These findings indicate that splicing variant involving exons 7 of IRF-1 could potentially modulate anti-angiogenic effect of IRF-1. In overall, this study provides the first evidence for anti-angiogenic role of IRF-1, which may have therapeutic values for cancer and angiogenesis-associated diseases.

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Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration.

Publication Date: 2008 Apr 22 PMID: 18471988
Authors: Yu, H. - Lee, I. - Salomon, A. R. - Yu, K. - Huttemann, M.
Journal: Biochim Biophys Acta

Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.

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Regulatory interactions in the dimeric cytochrome bc(1) complex: The advantages of being a twin.

Publication Date: 2008 Apr 22 PMID: 18471987
Authors: Covian, R. - Trumpower, B. L.
Journal: Biochim Biophys Acta

The dimeric cytochrome bc(1) complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc(1) complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.

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Identification of cold-inducible microRNAs in plants by transcriptome analysis.

Publication Date: 2008 Apr 18 PMID: 18471443
Authors: Zhou, X. - Wang, G. - Sutoh, K. - Zhu, J. K. - Zhang, W.
Journal: Biochim Biophys Acta

MicroRNAs are ~ 21-nt long, non-coding RNAs that play critical roles in post-transcriptional gene regulation. Even though a large number of miRNAs have been identified, annotating their functions remains a challenge. We develop a computational, transcriptome-based approach to annotating stress-inducible microRNAs in plants. With this approach, we find that nineteen microRNA genes of eleven microRNA families in Arabidopsis thaliana are up-regulated by cold stress. Our experiments validate that among the eleven microRNAs, eight are differentially induced and three are constantly expressed under low temperature. Our result expands the number of cold-inducible microRNAs from four to eight. A promoter analysis further reveals that the cold-responsive microRNA genes contain many known stress-related cis-regulatory elements in their promoters. Our analysis also indicates that many signaling pathways, such as auxin pathways, may be affected by cold-inducible microRNAs. Our approach can be applied to plant microRNAs responding to other abiotic and biotic stresses. The research demonstrates that machine learning methods, augmented by wet-lab analysis, hold a great promise for functional annotation of microRNAs.

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Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

Publication Date: 2008 Apr 18 PMID: 18471442
Authors: Qureshi, H. Y. - Ricci, G. - Zafarullah, M.
Journal: Biochim Biophys Acta

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

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Interrelated influence of superoxides and free fatty acids over mitochondrial uncoupling in skeletal muscle.

Publication Date: 2008 Apr 18 PMID: 18471434
Authors: Lombardi, A. - Grasso, P. - Moreno, M. - de Lange, P. - Silvestri, E. - Lanni, A. - Goglia, F.
Journal: Biochim Biophys Acta

Mitochondrial uncoupling protein 3 (UCP(3))-mediated uncoupling has been postulated to depend on several factors, including superoxides, free fatty acids (FFAs), and fatty acid hydroperoxides and/or their derivatives. We investigated whether there is an interrelation between endogenous mitochondrial superoxides and fatty acids in inducing skeletal muscle mitochondrial uncoupling, and we speculated on the possible involvement of UCP(3) in this process. In the absence of FFAs, no differences in proton-leak kinetic were detected between succinate-energized mitochondria respiring in the absence or presence of rotenone, despite a large difference in complex I superoxide production. The addition of either arachidic acid or arachidonic acid induced an increase in proton-leak kinetic, with arachidonic acid having the more marked effect. The uncoupling effect of arachidic acid was independent of the presence of GDP, rotenone and vitamin E, while that of arachidonic acid was dependent on these factors. These data demonstrate that FFA and O(2-) play interrelated roles in inducing mitochondrial uncoupling, and we hypothesize that a likely formation of mitochondrial fatty acid hydroperoxides is a key event in the arachidonic acid-induced GDP-dependent inhibition of mitochondrial uncoupling.

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Mitochondrial uncoupling protein 1 expression in thymocytes.

Publication Date: 2008 Apr 18 PMID: 18471433
Authors: Adams, A. E. - Carroll, A. M. - Fallon, P. G. - Porter, R. K.
Journal: Biochim Biophys Acta

Using a peptide antibody specific and selective to UCP 1, this study confirms the observation that mitochondrial uncoupling protein 1 (UCP 1) is present in thymocytes isolated from UCP 1 wild-type, but not UCP 1 knock-out mice. UCP 1 is also shown to be present in thymocytes isolated from rat. It was also demonstrated that an antibody raised to the full-length UCP 1 protein appears to be non-specific for UCP 1, as it detects protein in UCP 1 wild-type and UCP 1 knock-out mice, protein in mitochondria isolated from brown adipose tissue of both UCP 1 wild-type and UCP 1 knock-out mice, as well as detecting protein in mitochondria isolated from rat spleen, kidney, skeletal muscle and liver, tissues that do not express UCP 1. We were also able to show that CIDEA, a soluble protein with a suggested role in regulating UCP 1 function, is equally abundant in thymocytes from UCP 1 wild-type and UCP 1 knock-out mice. Taken together our data demonstrate that (a) UCP 1 is present in rat and mouse thymocytes, (b) that the antibody to full-length UCP 1 is not specific for UCP 1 and (c) that the absence of UCP 1 does not affect native expression of CIDEA in thymocytes.

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NADH/NAD(+) interaction with NADH: Ubiquinone oxidoreductase (complex I).

Publication Date: 2008 Apr 18 PMID: 18471432
Authors: Vinogradov, A. D.
Journal: Biochim Biophys Acta

The quantitative data on the binding affinity of NADH, NAD(+), and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.

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Mechanisms underlying the loss of mitochondrial membrane potential in glutamate excitotoxicity.

Publication Date: 2008 Apr 18 PMID: 18471431
Authors: Abramov, A. Y. - Duchen, M. R.
Journal: Biochim Biophys Acta

Glutamate excitotoxicity amplifies neuronal death following stroke. We have explored the mechanisms underlying the collapse of mitochondrial potential (Deltapsi(m)) and loss of [Ca(2+)](c) homeostasis in rat hippocampal neurons in culture following toxic glutamate exposure. The collapse of Deltapsi(m) is multiphasic and Ca(2+)-dependent. Glutamate induced a decrease in NADH autofluorescence which preceded the loss of Deltapsi(m). Both the decrease in NADH signal and the loss of Deltapsi(m) were suppressed by Ru360 and both were delayed by inhibition of PARP (by 3-AB or DPQ). During this period, addition of mitochondrial substrates (methyl succinate and TMPD-ascorbate) or buffering [Ca(2+)](i) (using BAPTA-AM or EGTA-AM), rescued Deltapsi(m). These data suggest that mitochondrial Ca(2+) uptake activates PARP which in turn depletes NADH, promoting the initial collapse of Deltapsi(m). After >~20 min, buffering Ca(2+) or substrate addition failed to restore Deltapsi(m). In neurons from cyclophilin D-/- (cypD-/-) mice or in cells treated with cyclosporine A, removal of Ca(2+) restored Deltapsi(m) even after 20 min of glutamate exposure, suggesting involvement of the mPTP in the irreversible depolarisation seen in WT cells. Thus, mitochondrial depolarisation represents two consecutive but distinct processes driving cell death, the first of which is reversible while the second is not.

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Determination of the rate of K(+) movement through potassium channels in isolated rat heart and liver mitochondria.

Publication Date: 2008 Apr 18 PMID: 18471430
Authors: Bednarczyk, P. - Barker, G. D. - Halestrap, A. P.
Journal: Biochim Biophys Acta

Both ATP-regulated (mitoK(ATP)) and large conductance calcium-activated (mitoBK(Ca)) potassium channels have been proposed to regulate mitochondrial K(+) influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K(+)-ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K(+)/H(+) exchanger nigericin. This allowed rates of valinomycin-induced K(+) influx to be determined whilst parallel measurements of light scattering (A(520)) and matrix volume ((3)H(2)O and [(14)C]-sucrose) enabled rates of K(+) influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K(+) influx of <5 nmol K(+) min(-1) per mg protein corresponding to <2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoK(ATP) channel openers or blockers. However, the mitoBK(Ca) opener NS1619 (10-50 microM) did decrease light scattering slightly, but this was also seen in K(+)-free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBK(Ca) blocker paxilline (10-50 microM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoK(ATP) or the mitoBK(Ca) channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.

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Distance metrics for heme protein electron tunneling.

Publication Date: 2008 Apr 18 PMID: 18471429
Authors: Moser, C. C. - Chobot, S. E. - Page, C. C. - Dutton, P. L.
Journal: Biochim Biophys Acta

There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 A longer. In a typical protein medium, such a distance difference normally corresponds to a ~1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.

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Determination of torque generation from the power stroke of Escherichia coli F(1)-ATPase.

Publication Date: 2008 Apr 18 PMID: 18471428
Authors: Hornung, T. - Ishmukhametov, R. - Spetzler, D. - Martin, J. - Frasch, W. D.
Journal: Biochim Biophys Acta

The torque generated by the power stroke of Escherichia coli F(1)-ATPase was determined as a function of the load from measurements of the velocity of the gamma-subunit obtained using a 0.25 micros time resolution and direct measurements of the drag from 40 to 95 nm gold nanorods. This result was compared to values of torque calculated using four different drag models. Although the gamma-subunit was able to rotate with a 20x increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63+/-8 pN nm, independent of the load on the enzyme.

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The effect of sulfhydryl groups and disulphide linkage in the thermal aggregation of Z19 alpha-zein.

Publication Date: 2008 May 5 PMID: 18466780
Authors: Cabra, V. - Vazquez-Contreras, E. - Moreno-Carcamo, A. - Arreguin-Espinosa, R.
Journal: Biochim Biophys Acta

Zeins are the major storage proteins in corn seeds organized in protein bodies located in the endosperm. They are soluble in alcoholic solution and depict a high tendency to aggregation. The Z19 alpha-zein aggregates obtained by heating show a particular and interesting temperature-dependent behavior. This work was aimed at determining not only the effect of temperature on the aggregation behavior, but also the effect of the sulfhydryl groups and disulphide bonds on the thermal aggregation process under non-aqueous conditions. Z19 alpha-zein was chemically modified to obtain different sulfhydryl groups and disulphide-bonds content. Far-UV CD, ANS emission fluorescence, and dynamic light scattering, as well as differential scanning calorimetry, were performed to characterize this protein. Removal of these disulphide-bonds and alkylation of all the sulfhydryl groups in the protein promoted the lowest T(m) of 57.36 degrees C, eliminated aggregation, enhanced protein flexibility, and diminished thermal stability. These results suggest that the disulphide linkage could be the driving force for the Z19 alpha-zein aggregation.

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Sequence variation of Vanabin2-like vanadium-binding proteins in blood cells of the vanadium-accumulating ascidian Ascidia sydneiensis samea.

Publication Date: 2008 May 5 PMID: 18466774
Authors: Ueki, T. - Satake, M. - Kamino, K. - Michibata, H.
Journal: Biochim Biophys Acta

The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1-4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea. In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal-ion selectivity, but the overall affinity of 14MT for VO(2+) is higher than that of Vanabin2. Binding number for VO(2+) ions was the same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high-affinity binding sites for VO(2+) are conserved among Vanabin2 variants.

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Nitrotyrosine promotes human aortic smooth muscle cell migration through oxidative stress and ERK1/2 activation.

Publication Date: 2008 May 3 PMID: 18460343
Authors: Mu, H. - Wang, X. - Lin, P. - Yao, Q. - Chen, C.
Journal: Biochim Biophys Acta

Nitrotyrosine is a new biomarker of atherosclerosis and inflammation. The objective of this study was to determine the direct effects of free nitrotyrosine on human aortic smooth muscle cell (AoSMC) migration and molecular mechanisms. By a modified Boyden chamber assay, nitrotyrosine significantly increased AoSMC migration in a concentration-dependent manner. For example, nitrotyrosine at 300 nM increased AoSMC migration up to 152% compared with l-tyrosine-treated control cells (P<0.01). Cell wound healing assay confirmed this effect. Nitrotyrosine significantly increased the expression of some key cell migration-related molecules including PDGF receptor-B, matrix metalloproteinase 2 (MMP2) and integrins alphaV and beta3 at both mRNA and protein levels in AoSMC (P<0.01). In addition, nitrotyrosine increased reactive oxygen species (ROS) production in AoSMC by staining with fluorescent dye DCFHDA. Furthermore, nitrotyrosine induced transient phosphorylation of ERK2 by Bio-Plex luminex immunoassay and western blot analysis. AoSMC were able to uptake nitrotyrosine. Antioxidants including seleno-l-methionine and superoxide dismutase mimetic (MnTBAP) as well as ERK1/2 inhibitor PD98059 effectively blocked the promoting effect of nitrotyrosine on AoSMC migration and the mRNA expression of above cell migration-related molecules. Thus, nitrotyrosine directly increases AoSMC migration in vitro and the expression of migration-related molecules through overproduction of ROS and activation of ERK1/2 pathway. Nitrotyrosine may contribute to cardiovascular pathogenesis.

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Structure, function and interactions of the PufX protein.

Publication Date: 2008 Apr 18 PMID: 18460337
Authors: Holden-Dye, K. - Crouch, L. I. - Jones, M. R.
Journal: Biochim Biophys Acta

The PufX protein is an important component of the reaction centre-light-harvesting 1 (RC-LH1) complex of Rhodobacter species of purple photosynthetic bacteria. Early studies showed that removal of the PufX protein causes changes in the structure of the RC-LH1 complex that result in a loss of the capacity for photosynthetic growth, and that this loss can be overcome though further mutations that change the structure of the LH1 antenna. More recent studies have examined interactions of the PufX protein with other components of the RC-LH1 complex. This review considers our current understanding of the structure and function of the PufX protein, how this protein interacts with other components of the photosynthetic membrane, and its influence on the oligomeric state of the RC-LH1 complex and the larger-scale architecture of the photosynthetic membrane.

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The role of microRNAs and other endogenous small RNAs in plant stress responses.

Publication Date: 2008 Apr 16 PMID: 18457682
Authors: Shukla, L. I. - Chinnusamy, V. - Sunkar, R.
Journal: Biochim Biophys Acta

Crop yields are significantly reduced by biotic and abiotic stresses throughout the world. To reduce the damage caused by stress factors, plants have evolved sophisticated adaptive responses involving reprogramming gene expression at the transcriptional, post-transcriptional and post-translational levels. A better understanding of such processes will lead to new strategies to improve plant stress tolerance. Recently discovered endogenous small RNAs (microRNAs and small-interfering RNAs) have emerged as important players in plant stress responses. The observation that some of the small RNAs are up- or down-regulated in response to stress implies that these small RNAs have a role in stress tolerance. Stress-induced small RNAs might down-regulate their target genes, which may encode negative regulators of stress responses. Conversely, small RNAs down-regulated in response to stress cause the accumulation of their target mRNAs, which may contribute positively to the adaptation to stress. Here, we review the current status of small RNAs involved in biotic and abiotic stress regulatory networks.

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Peroxidase activity of hemoglobin towards ascorbate and urate: A synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC).

Publication Date: 2008 Apr 16 PMID: 18457681
Authors: Cooper, C. E. - Silaghi-Dumitrescu, R. - Rukengwa, M. - Alayash, A. I. - Buehler, P. W.
Journal: Biochim Biophys Acta

Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties ("blood substitutes") are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a pK(a) of 4.7, consistent with protonation of the ferryl species (Fe(IV)OH(-)) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.

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Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and DeltaF508 CFTR.

Publication Date: 2008 Apr 16 PMID: 18457676
Authors: Okiyoneda, T. - Niibori, A. - Harada, K. - Kohno, T. - Michalak, M. - Duszyk, M. - Wada, I. - Ikawa, M. - Shuto, T. - Suico, M. A. - Kai, H.
Journal: Biochim Biophys Acta

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.

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A p38-p65 transcription complex induced by endothelin-1 mediates signal transduction in cancer cells.

Publication Date: 2008 Apr 16 PMID: 18457675
Authors: von Brandenstein, M. G. - Ngum Abety, A. - Depping, R. - Roth, T. - Koehler, M. - Dienes, H. P. - Fries, J. W.
Journal: Biochim Biophys Acta

Endothelin-1 is a powerful mitogen for various tumor and non-tumor cells. Its signaling cascade induces the inflammatory NF-kappaB complex, leading to expression of a number of target genes. In this context, MAPK p38 has been regarded as a potential phosphate donor for the p65 subunit of NF-kappaB. In the present study in HeLa cells, we have found that ET-1 induced signalling activates the NF-kappaB transcription complex (TC) in the nucleus at 6 h specifically via ET-A - but not ET-B receptor. The TC contains p65, p38 (alpha and beta) - binding to the NLS of p65 in the cytoplasm - as well as p50, but no IkappaBalpha. Specific p38 inhibition by SB203580 or by siRNA interferes markedly with gene expression of several target genes. Complex formation occurs in the cytoplasm, and both transcription factors transmigrate as a complex in the nucleus. Overexpression of p38, treatment with Chrysin, MG132, or dimethylformamide shows dependence of TC on p38 as partner. In other tumor cells lines studied, ET-1 activates TC, with p38 as an important complex partner of p65. TC-induction by ET-1 contains about twice the amount of p38 than by TNFalpha. Thus, p38 may be an additional therapeutic target to control inflammatory gene expression in tumor cells.

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Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway.

Publication Date: 2008 Apr 16 PMID: 18457654
Authors: Lepp, H. - Salomonsson, L. - Zhu, J. P. - Gennis, R. B. - Brzezinski, P.
Journal: Biochim Biophys Acta

Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O(2) to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O(2) reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O(2) reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O(2) reduction, but a decrease in the apparent pK(a) of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.

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The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen.

Publication Date: 2008 Apr 16 PMID: 18456010
Authors: Krarup, A. - Gulla, K. C. - Gal, P. - Hajela, K. - Sim, R. B.
Journal: Biochim Biophys Acta

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and that its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.

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Antiplasmin activity of natural occurring polyphenols.

Publication Date: 2008 Apr 16 PMID: 18456009
Authors: Mozzicafreddo, M. - Cuccioloni, M. - Bonfili, L. - Eleuteri, A. M. - Fioretti, E. - Angeletti, M.
Journal: Biochim Biophys Acta

The equilibrium between proteolytic enzymes and their cognate inhibitors is crucial in a number of physiological as well as pathological processes, including cancer, inflammatory processes and thrombosis. Therefore, both synthetic and natural small molecule inhibitors are object of extensive studies as drugs in the treatment of these pathologies. Two natural occurring polyphenolic compounds, representative of glycosylated and unglycosylated flavonoid structures, namely quercetin and rutin, were thereby tested as potential ligands of plasmin(ogen), a serine (pro)protease, whose role in tumor cell invasion and migration has been reported. Quercetin showed a ten folds higher affinity with plasmin with respect to rutin in terms of equilibrium dissociation constant, both compounds acting as in vitro moderate reversible inhibitors; additionally, quercetin and rutin prevented plasmin-incubated BB1 cells from releasing E-cadherin fragment to a different extent, respectively. Furthermore, a feasible mechanism of interaction was analyzed and discussed using a molecular modeling approach.

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alpha-Enolase binds to human plasminogen on the surface of Bacillus anthracis.

Publication Date: 2008 Apr 16 PMID: 18456007
Authors: Agarwal, S. - Kulshreshtha, P. - Bambah Mukku, D. - Bhatnagar, R.
Journal: Biochim Biophys Acta

alpha-enolase of Bacillus anthracis has recently been classified as an immunodominant antigen and a potent virulence factor determinant. alpha-enolase (2-phospho-d-glycerate hydrolase (EC 4.2.1.11), a key glycolytic metalloenzyme catalyzes the dehydration of d-(+)-2-phosphoglyceric acid to phosphoenolpyruvate. Interaction of surface bound alpha-enolase with plasminogen has been incriminated in tissue invasion for pathogenesis. B. anthracis alpha-enolase was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity that exhibited a K(m) of 3.3 mM for phosphoenolpyruvate and a V(max) of 0.506 microMmin(- 1) mg(- 1). B. anthracis whole cells and membrane vesicles probed with anti-enolase antibodies confirmed the surface localization of alpha-enolase. The specific interaction of alpha-enolase with human plasminogen (but not plasmin) evident from ELISA and the retardation in the native gel reinforced its role in plasminogen binding. Putative plasminogen receptors in B. anthracis other than enolase were also observed. This binding was found to be carboxypeptidase sensitive implicating the role of C-terminal lysine residues. The recombinant enolase displayed in vitro laminin binding, an important mammalian extracellular matrix protein. Plasminogen interaction conferred B. anthracis with a potential to in vitro degrade fibronectin and exhibit fibrinolytic phenotype. Therefore, by virtue of its interaction to host plasminogen and extracellular matrix proteins, alpha-enolase may contribute in augmenting the invasive potential of B. anthracis.

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Conformation, thermodynamics and stoichiometry of HSA adsorbed to colloidal CdSe/ZnS quantum dots.

Publication Date: 2008 Apr 16 PMID: 18456006
Authors: Xiao, Q. - Huang, S. - Qi, Z. D. - Zhou, B. - He, Z. K. - Liu, Y.
Journal: Biochim Biophys Acta

Water-soluble luminescent colloidal quantum dots (QDs) have attracted great attention in biological and medical applications. In particular, for any potential in vivo application, the interaction of QDs with human serum albumin (HSA) is crucial. As a step toward the elucidation of the fate of QDs introduced to organism, the interactions between QDs and HSA were systematically investigated by various spectroscopic techniques under the physiological conditions. It was proved that binding of QDs and HSA is a result of the formation of QDs-HSA complex and electrostatic interactions play a major role in stabilizing the complex. The modified Stern-Volmer quenching constant K(a) at different temperatures and corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. Furthermore, the site marker competitive experiments revealed that the binding location of QDs with HSA is around site I, centered at Lys199. The conformational changes of HSA induced by QDs have been analyzed by means of CD and FT-IR. The results suggested that HSA underwent substantial conformational changes at both secondary and tertiary structure levels. The stoichiometry of HSA attached to QDs was obtained by dynamic light scattering (DLS) and xi-potential.

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Diphenyleneiodonium acutely inhibits reactive oxygen species production by mitochondrial complex I during reverse, but not forward electron transport.

Publication Date: 2008 May PMID: 18395512
Authors: Lambert, A. J. - Buckingham, J. A. - Boysen, H. M. - Brand, M. D.
Journal: Biochim Biophys Acta

We investigated the effects of diphenyleneiodonium (DPI) on superoxide production by complex I in mitochondria isolated from rat skeletal muscle. Superoxide production was measured indirectly as hydrogen peroxide production. In a conventional medium containing chloride, DPI strongly inhibited superoxide production by complex I driven by reverse electron transport from succinate. In principle, this inhibition could be explained by an observed decrease in the mitochondrial pH gradient caused by the known chloride-hydroxide antiport activity of DPI. In a medium containing gluconate instead of chloride, DPI did not affect the pH gradient. In this gluconate medium, DPI still inhibited superoxide production driven by reverse electron transport, showing that the inhibition of superoxide production was not dependent on changes in the pH gradient. It had no effect on superoxide production during forward electron transport from NAD-linked substrates in the presence of rotenone (to maximise superoxide production from the flavin of complex I) or antimycin (to maximise superoxide production from complex III), suggesting that the effects of DPI were not through inhibition of the flavin. We conclude that DPI has the novel and potentially very useful ability to prevent superoxide production from the site in complex I that is active during reverse electron transport, without affecting superoxide production during forward electron transport.

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Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions.

Publication Date: 2008 May PMID: 18394424
Authors: Reinhold, C. - Niczyporuk, S. - Beran, K. C. - Jahns, P.
Journal: Biochim Biophys Acta

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.

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The energetics of the primary proton transfer in bacteriorhodopsin revisited: It is a sequential light-induced charge separation after all.

Publication Date: 2008 May PMID: 18387356
Authors: Braun-Sand, S. - Sharma, P. K. - Chu, Z. T. - Pisliakov, A. V. - Warshel, A.
Journal: Biochim Biophys Acta

The light-induced proton transport in bacteriorhodopsin has been considered as a model for other light-induced proton pumps. However, the exact nature of this process is still unclear. For example, it is not entirely clear what the driving force of the initial proton transfer is and, in particular, whether it reflects electrostatic forces or other effects. The present work simulates the primary proton transfer (PT) by a specialized combination of the EVB and the QCFF/PI methods. This combination allows us to obtain sufficient sampling and a quantitative free energy profile for the PT at different protein configurations. The calculated profiles provide new insight about energetics of the primary PT and its coupling to the protein conformational changes. Our finding confirms the tentative analysis of an earlier work (A. Warshel, Conversion of light energy to electrostatic energy in the proton pump of Halobacterium halobium, Photochem. Photobiol. 30 (1979) 285-290) and determines that the overall PT process is driven by the energetics of the charge separation between the Schiff base and its counterion Asp85. Apparently, the light-induced relaxation of the steric energy of the chromophore leads to an increase in the ion-pair distance, and this drives the PT process. Our use of the linear response approximation allows us to estimate the change in the protein conformational energy and provides the first computational description of the coupling between the protein structural changes and the PT process. It is also found that the PT is not driven by twist-modulated changes of the Schiff base's pK(a), changes in the hydrogen bond directionality, or other non-electrostatic effects. Overall, based on a consistent use of structural information as the starting point for converging free energy calculations, we conclude that the primary event should be described as a light-induced formation of an unstable ground state, whose relaxation leads to charge separation and to the destabilization of the ion-pair state. This provides the driving force for the subsequent PT steps.

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Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.

Publication Date: 2008 May PMID: 18373974
Authors: Mahammad, S. - Parmryd, I.
Journal: Biochim Biophys Acta

Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [(3)H]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.

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Thermodynamic analysis of l-arginine and N(omega)-hydroxy-l-arginine binding to nitric oxide synthase.

Publication Date: 2008 May PMID: 18371313
Authors: Zakariassen, H. - Cederkvist, F. H. - Harbitz, E. - Shimizu, T. - Lange, R. - Mayer, B. - Gorren, A. C. - Andersson, K. K. - Sorlie, M.
Journal: Biochim Biophys Acta

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-l-arginine (NHA) has a larger affinity than l-Arginine (l-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and l-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to l-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and l-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.

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Feedback regulation of photosynthetic electron transport by NADP(H) redox poise.

Publication Date: 2008 May PMID: 18371296
Authors: Hald, S. - Nandha, B. - Gallois, P. - Johnson, G. N.
Journal: Biochim Biophys Acta

When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process - feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines - one having reduced levels of ferredoxin NADP(+)-reductase (FNR), the enzyme responsible for reducing NADP(+), and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.

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Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases.

Publication Date: 2008 May PMID: 18371295
Authors: de Oliveira, H. C. - Wulff, A. - Saviani, E. E. - Salgado, I.
Journal: Biochim Biophys Acta

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O(2)(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O(2)(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O(2)(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O(2)(-) production were stimulated by free Ca(2+) concentration. Together, these results indicate that Ca(2+)-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O(2)(-) production that favors NO degradation in potato tuber mitochondria.

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Generation of reactive oxygen species upon strong visible light irradiation of isolated phycobilisomes from Synechocystis PCC 6803.

Publication Date: 2008 May PMID: 18371294
Authors: Rinalducci, S. - Pedersen, J. Z. - Zolla, L.
Journal: Biochim Biophys Acta

The mechanism of photodegradation of antenna system in cyanobacteria was investigated using spin trapping ESR spectroscopy, SDS-PAGE and HPLC-MS. Exposure of isolated intact phycobilisomes to illumination with strong white light (3500 mumol m(-2) s(-1) photosynthetically active radiation) gave rise to the formation of free radicals, which subsequently led to specific protein degradation as a consequence of reactive oxygen species-induced cleavage of the polypeptide backbone. The use of specific scavengers demonstrated an initial formation of both singlet oxygen ((1)O(2)) and superoxide (O(2)(-)), most likely after direct reaction of molecular oxygen with the triplet state of phycobiliproteins, generated from intersystem crossing of the excited singlet state. In a second phase carbon-based radicals, detected through the appearance of DMPO-R() adducts, were produced either via O(2)(-) or by direct (1)O(2) attack on amino acid moieties. Thus photo-induced degradation of intact phycobilisomes in cyanobacteria occurs through a complex process with two independent routes leading to protein damage: one involving superoxide and the other singlet oxygen. This is in contrast to the mechanism found in plants, where damage to the light-harvesting complex proteins has been shown to be mediated entirely by (1)O(2) generation.

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Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2.

Publication Date: 2008 May PMID: 18359306
Authors: Ahmed, S. - Kapoor, D. - Singh, B. - Guptasarma, P.
Journal: Biochim Biophys Acta

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.

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Dynamics of oligomer formation by denatured carbonic anhydrase II.

Publication Date: 2008 May PMID: 18359304
Authors: Prokhorov, D. A. - Timchenko, A. A. - Uversky, V. N. - Khristoforov, V. S. - Kihara, H. - Kimura, K. - Kutyshenko, V. P.
Journal: Biochim Biophys Acta

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.

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Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates.

Publication Date: 2008 May PMID: 18359303
Authors: Lee, I. - Suzuki, C. K.
Journal: Biochim Biophys Acta

Lon, also known as the protease La, is a homo-oligomeric ATP-dependent protease, which is highly conserved in archaea, eubacteria and eukaryotic mitochondria and peroxisomes. Since its discovery, studies have shown that Lon activity is essential for cellular homeostasis, mediating protein quality control and metabolic regulation. This article highlights the discoveries made over the past decade demonstrating that Lon selectively degrades abnormal as well as certain regulatory proteins and thus plays significant roles in maintaining bacterial and mitochondrial function and integrity. In addition, Lon is required in certain pathogenic bacteria, for rendering pathogenicity and host infectivity. Recent research endeavors have been directed toward elucidating the reaction mechanism of the Lon protease by different biochemical and structural biological techniques. In this mini-review, the authors survey the diverse biological roles of Lon, and also place special emphasis on recent findings that clarify the mechanistic aspects of the Lon reaction cycle.

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Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1.

Publication Date: 2008 May PMID: 18359302
Authors: Ekhlasi-Hundrieser, M. - Calvete, J. J. - Von Rad, B. - Hettel, C. - Nimtz, M. - Topfer-Petersen, E.
Journal: Biochim Biophys Acta

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.

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The role of the sulfonium linkage in the stabilization of the ferrous form of myeloperoxidase: A comparison with lactoperoxidase.

Publication Date: 2008 May PMID: 18359301
Authors: Brogioni, S. - Stampler, J. - Furtmuller, P. G. - Feis, A. - Obinger, C. - Smulevich, G.
Journal: Biochim Biophys Acta

In all mammalian peroxidases, the heme is covalently attached to the protein via two ester linkages between conserved aspartate (Asp94) and glutamate residues (Glu242) and modified methyl groups on pyrrole rings A and C. Only myeloperoxidase has an additional sulfonium ion linkage between the sulfur atom of the conserved methionine 243 and the beta-carbon of the vinyl group on pyrrole ring A. Upon reduction from Fe(III) to Fe(II), lactoperoxidase (LPO) but not myeloperoxidase (MPO) is shown to adopt three distinct active site conformations which depend on pH and time. Comparative spectroscopic analysis (UV-Vis absorption and resonance Raman) of the ferrous forms of LPO, wild-type MPO and the variants Asp94Val, Glu242Gln, Met243Thr and Met243Val clearly demonstrate that a single, stable ferrous form of MPO is present only in those proteins which retain an intact sulfonium linkage. By contrast, both ferrous Met243Thr and Met243Val can assume two conformations. They resemble ferrous LPO, being five-coordinated high-spin species that are distinguished by the strength of the proximal Fe-histidine bond. This bond weakens with time or decreasing pH, as indicated by the Fe-histidine stretching bands.

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Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning.

Publication Date: 2008 May PMID: 18359300
Authors: Teramoto, R. - Minagawa, H. - Honda, M. - Miyazaki, K. - Tabuse, Y. - Kamijo, K. - Ueda, T. - Kaneko, S.
Journal: Biochim Biophys Acta

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high alpha-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

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A Kunitz trypsin inhibitor of Entada scandens seeds: Another member with single disulfide bridge.

Publication Date: 2008 May PMID: 18359299
Authors: Lingaraju, M. H. - Gowda, L. R.
Journal: Biochim Biophys Acta

Sword bean (Entada scandens) is a tree climber that belongs to Mimosoideae, a subfamily of Leguminosae. A potent Kunitz type trypsin inhibitor (ESTI) was purified to homogeneity from Entada scandens seeds by sequential ammonium sulfate precipitation, affinity chromatography on trypsin-Sepharose and DEAE-Sephacel ion-exchange chromatography. ESTI is a single polypeptide chain of 19,766 Da. Both native PAGE as well as isoelectric focusing showed a single inhibitor species with a pI of 7.43. MALDI-TOF analysis also confirmed the monomeric nature. The amino-terminal sequence of ESTI reveals significant homology to the Kunitz-type protease inhibitors of legume plants. ESTI is unique in that it contains a single disulfide bridge, and unlike other inhibitors from Mimosoideae species is a single chain polypeptide. ESTI inhibited bovine trypsin with a stoichiometry of 1:1 and the apparent K(i) was 4.9x10(-9) M. In vitro assay showed that ESTI inhibited the midgut proteinase of the fifth instar larvae of Rice moth (Corcyra cephalonica) with an IC(50) of 26.4+/-0.01 nM. ESTI exhibits a mixed type competitive inhibition at lower concentration and pure competitive at higher inhibitor concentrations. Phylogenetic analyses depicted a clear divergence of single disulfide containing inhibitors from other tree legume Kunitz inhibitors. The homology of ESTI to Kunitz inhibitors together with the absence of Bowman-Birk type inhibitors in sword bean further supports the theory that there exists an evolutionary relationship between the families of inhibitors found in Leguminosae.

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Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4.

Publication Date: 2008 May PMID: 18359284
Authors: Liu, J. - Krulwich, T. A. - Hicks, D. B.
Journal: Biochim Biophys Acta

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na(+)/H(+) antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na(+)-resistance on an antiporter-deficient E. coli strain, nor did they confer Na(+)/H(+) antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.

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Binding of phospholipids to beta-Lactoglobulin and their transfer to lipid bilayers.

Publication Date: 2008 May PMID: 18358232
Authors: Martins, P. A. - Gomes, F. - Vaz, W. L. - Moreno, M. J.
Journal: Biochim Biophys Acta

The bovine milk lipocalin, beta-Lactoglobulin (beta-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC(10)PE) to beta-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K(B), was (1.2+/-0.2)x10(6) M(-1) at 25 degrees C, pH 7.4 and I=0.15 M. Dependence of K(B) on pH and on the monomer-dimer equilibrium of beta-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the beta-barrel in the protein. The monomer-dimer equilibrium of beta-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K(D), was (7.0+/-1.5)x10(5) M(-1) at 25 degrees C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC(10)PE between beta-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. beta-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.

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Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae.

Publication Date: 2008 May PMID: 18355437
Authors: Kamp, C. - Silakov, A. - Winkler, M. - Reijerse, E. J. - Lubitz, W. - Happe, T.
Journal: Biochim Biophys Acta

Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C. reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 mumol H(2) min(-1) mg(-1) was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the H(ox)-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand.

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Determination of the excitation migration time in Photosystem II Consequences for the membrane organization and charge separation parameters.

Publication Date: 2008 May PMID: 18355436
Authors: Broess, K. - Trinkunas, G. - van Hoek, A. - Croce, R. - van Amerongen, H.
Journal: Biochim Biophys Acta

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rogner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].

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Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign.

Publication Date: 2008 May PMID: 18346474
Authors: Host, G. E. - Jonsson, B. H.
Journal: Biochim Biophys Acta

Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occurring esterases. The design was based on a previously developed strategy [G. Host, L.G. Martensson, B.H. Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, Biochim. Biophys. Acta 1764 (2006) 1601-1606.], in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with k(cat)/K(M)=625 (+/- 38) M(-1) s(-1). It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with k(cat)/K(M)=101,700 (+/- 4800) M(-1) s(-1), which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable K(M) values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV), but for V121A/V143A/T200A no K(M) could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, k(cat)/K(M) is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.

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Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver.

Publication Date: 2008 May PMID: 18346472
Authors: Koo, H. Y. - Wallig, M. A. - Chung, B. H. - Nara, T. Y. - Cho, B. H. - Nakamura, M. T.
Journal: Biochim Biophys Acta

Dietary fructose has been suspected to contribute to development of metabolic syndrome. However, underlying mechanisms of fructose effects are not well characterized. We investigated metabolic outcomes and hepatic expression of key regulatory genes upon fructose feeding under well defined conditions. Rats were fed a 63% (w/w) glucose or fructose diet for 4 h/day for 2 weeks, and were killed after feeding or 24-hour fasting. Liver glycogen was higher in the fructose-fed rats, indicating robust conversion of fructose to glycogen through gluconeogenesis despite simultaneous induction of genes for de novo lipogenesis and increased liver triglycerides. Fructose feeding increased mRNA of previously unidentified genes involved in macronutrient metabolism including fructokinase, aldolase B, phosphofructokinase-1, fructose-1,6-bisphosphatase and carbohydrate response element binding protein (ChREBP). Activity of glucose-6-phosphate dehydrogenase, a key enzyme for ChREBP activation, remained elevated in both fed and fasted fructose groups. In the fasted liver, the fructose group showed lower non-esterified fatty acids, triglycerides and microsomal triglyceride transfer protein mRNA, suggesting low VLDL synthesis even though plasma VLDL triglycerides were higher. In conclusion, fructose feeding induced a broader range of genes than previously identified with simultaneous increase in glycogen and triglycerides in liver. The induction may be in part mediated by ChREBP.

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