Biochimica et Biophysica Acta

Small-molecule regulators that mimic transcription factors.

Publication Date: 2010 Aug 27 PMID: 20804876
Authors: Rodriguez-Martinez, J. A. - Peterson-Kaufman, K. J. - Ansari, A. Z.
Journal: Biochim Biophys Acta

Transcription factors (TFs) are responsible for decoding and expressing the information stored in the genome, which dictates cellular function. Creating artificial transcription factors (ATFs) that mimic endogenous TFs is a major goal at the interface of biology, chemistry, and molecular medicine. Such molecular tools will be essential for deciphering and manipulating transcriptional networks that lead to particular cellular states. In this minireview, the framework for the design of functional ATFs is presented and current challenges in the successful implementation of ATFs are discussed.

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Massive translational repression of gene expression during mouse erythroid differentiation.

Publication Date: 2010 Aug 27 PMID: 20804875
Authors: Pradet-Balade, B. - Leberbauer, C. - Schweifer, N. - Boulme, F.
Journal: Biochim Biophys Acta

We took advantage of a mouse erythroid differentiation system to determine the relative contribution of transcriptional and translational control during this process. Comparison of expression data obtained with total cytoplasmic mRNAs or polysome-bound mRNAs (actively translated mRNAs) on Affymetrix high-density oligonucleotide microarrays revealed different characteristics of the two regulatory mechanisms. Indeed, mRNA expression from a vast majority of genes was affected, albeit most changes were relatively small and occurred at a low pace. Translational control, however, affected a smaller fraction of genes but was effective at earlier time-points. This analysis unravels six clusters of genes showing no significant variation in mRNA expression levels whereas they are submitted to translational regulation. Their involvement in terminal mouse erythropoiesis may prove to be highly relevant. Furthermore, the data from specific and functional categories of genes emphasize that translational control, not only reinforces the transcriptional effect, but allows the cell to increase the complexity in gene expression regulation patterns.

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Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion.

Publication Date: 2010 Aug 27 PMID: 20804843
Authors: Hoang, M. V. - Smith, L. E. - Senger, D. R.
Journal: Biochim Biophys Acta

In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia. Mechanistically, these calpain inhibitors improved stability and organization of the actin cytoskeleton in retinal endothelial cells undergoing capillary morphogenesis in vitro, and they similarly improved organization of actin cables within new blood vessels in vivo. Hypoxia induced calpain activity in retinal endothelial cells and severely disrupted of the actin cytoskeleton, whereas calpain inhibitors preserved actin cables under hypoxic conditions. Collectively, these findings support the hypothesis that hyper-activation of calpains by hypoxia contributes to disruption of the retinal endothelial cell cytoskeleton, resulting in formation of neovessels that are defective both architecturally and functionally. Modest suppression of calpain activity with calpain inhibitors restores cytoskeletal architecture and promotes formation of a functional neovasculature, thereby reducing underlying hypoxia. In sharp contrast to "anti-angiogenesis" strategies that cannot restore normoxia and may aggravate hypoxia, the therapeutic strategy described here does not inhibit neovascularization. Instead, by improving the function of neovascularization to reduce underlying hypoxia, moderate calpain inhibition offers a method for alleviating retinal ischemia, thereby suggesting a new treatment paradigm based on improvement rather than inhibition of new blood vessel growth.

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Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique.

Publication Date: 2010 Aug 27 PMID: 20804726
Authors: Ohvo-Rekila, H. - Mattjus, P.
Journal: Biochim Biophys Acta

The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now for the first time analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane interacting proteins.

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Metabolic management of brain cancer.

Publication Date: 2010 Aug 27 PMID: 20804725
Authors: Seyfried, T. N. - Kiebish, M. A. - Marsh, J. - Shelton, L. M. - Huysentruyt, L. C. - Mukherjee, P.
Journal: Biochim Biophys Acta

Malignant brain tumors are a significant health problem in children and adults. Conventional therapeutic approaches have been largely unsuccessful in providing long-term management. As primarily a metabolic disease, malignant brain cancer can be managed through changes in metabolic environment. In contrast to normal neurons and glia, which readily transition to ketone bodies (beta-hydroxybutyrate) for energy under reduced glucose, malignant brain tumors are strongly dependent on glycolysis for energy. The transition from glucose to ketone bodies as a major energy source is an evolutionary conserved adaptation to food deprivation that permits the survival of normal cells during extreme shifts in nutritional environment. Only those cells with a flexible genome and normal mitochondria can effectively transition from one energy state to another. Mutations restrict genomic and metabolic flexibility thus making tumor cells more vulnerable to energy stress than normal cells. We propose an alternative approach to brain cancer management that exploits the metabolic flexibility of normal cells at the expense of the genetically defective and metabolically challenged tumor cells. This approach to brain cancer management is supported from recent studies in mice and humans treated with calorie restriction and the ketogenic diet. Issues of implementation and use protocols are presented for the metabolic management of brain cancer.

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The Warburg and Crabtree effects: On the origin of cancer cell energy metabolism and of yeast glucose repression.

Publication Date: 2010 Aug 27 PMID: 20804724
Authors: Diaz-Ruiz, R. - Rigoulet, M. - Devin, A.
Journal: Biochim Biophys Acta

During the last decades a considerable amount of research has been focused on cancer. Recently, tumor cell metabolism has been considered as a possible target for cancer therapy. It is widely accepted that tumors display enhanced glycolytic activity and impaired oxidative phosphorylation (Warburg effect). Therefore, it seems reasonable that disruption of glycolysis might be a promising candidate for specific anti-cancer therapy. Nevertheless, the concept of aerobic glycolysis as the paradigm of tumor cell metabolism has been challenged, as some tumor cells exhibit high rates of oxidative phosphorylation. Mitochondrial physiology in cancer cells is linked to the Warburg effect. Besides, its central role in apoptosis makes this organelle a promising "dual hit target" to selectively eliminate tumor cells. From a metabolic point of view, the fermenting yeast Saccharomyces cerevisiae and tumor cells share several features. In this paper we will review these common metabolic properties as well as the possible origins of the Crabtree and Warburg effects.

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Connexins: Sensors and regulators of cell cycling.

Publication Date: 2010 Aug 26 PMID: 20801193
Authors: Vinken, M. - Decrock, E. - De Vuyst, E. - Ponsaerts, R. - D'hondt, C. - Bultynck, G. - Ceelen, L. - Vanhaecke, T. - Leybaert, L. - Rogiers, V.
Journal: Biochim Biophys Acta

It is nowadays well established that gap junctions are critical gatekeepers of cell proliferation, by controlling the intercellular exchange of essential growth regulators. In recent years, however, it has become clear that the picture is not as simple as originally anticipated, as structural precursors of gap junctions can affect cell cycling by performing actions not related to gap junctional intercellular communication. Indeed, connexin hemichannels also foresee a pathway for cell growth communication, albeit between the intracellular compartment and the extracellular environment, while connexin proteins as such can directly or indirectly influence the production of cell cycle regulators independently of their channel activities. Furthermore, a novel set of connexin-like proteins, the pannexins, have lately joined in as regulators of the cell proliferation process, which they can affect as either single units or as channel entities. In the current paper, these multifaceted aspects of connexin-related signalling in cell cycling are reviewed.

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Hepcidin Expression by Human Monocytes in Response to Adhesion and Pro-Inflammatory Cytokines.

Publication Date: 2010 Aug 26 PMID: 20801192
Authors: Zhang, X. - Rovin, B. H.
Journal: Biochim Biophys Acta

BACKGROUND: A previous urine proteomic analysis from our laboratory suggested that hepcidin may be a biomarker for lupus nephritis flare. Immunohistochemical staining of kidney biopsies from lupus patients showed that hepcidin was expressed by infiltrating renal leukocytes. Here we investigated whether inflammatory cytokines relevant to the pathogenesis of lupus nephritis and other glomerular diseases regulate hepcidin expression by human monocytes. METHODS: Human CD14+ monocytes were incubated with interferon alpha (IFNalpha), interferon gamma (IFNgamma), interleukin-6 (IL6), interleukin-1 beta (IL1beta), monocyte chemotactic factor-1 (MCP1), or tumor necrosis factor alpha (TNFalpha). Hepcidin expression was examined by real-time PCR and enzyme immunoassay. RESULTS: Monocyte hepcidin mRNA increased during adherence to the tissue culture wells, reaching a level 150-fold higher than baseline within 12 hours of plating. After accounting for the effects of adhesion, monocytes showed time and dose-dependent up-regulation of hepcidin mRNA upon treatment with IFNalpha or IL6. One hour of incubation with IFNalpha or IL6 increased hepcidin mRNA 20 and 80-fold respectively; by 24 hours the mRNA remained 5 and 2.4-fold higher than baseline. IL1beta, IFNgamma, and MCP-1 did not affect monocyte hepcidin expression. TNFalpha inhibited hepcidin induction by IL6 in monocytes by 44%. After 24 hours of treatment with IFNalpha or IL6, immunoreactive hepcidin production by monocytes increased 3 and 2.6-fold respectively. CONCLUSION: Human monocytes produce hepcidin in response to adhesion and the pro-inflammatory cytokines IFNalpha and IL6. GENERAL SIGNIFICANCE: The appearance of hepcidin in the kidneys or urine during glomerular diseases may be from infiltrating monocytes induced to express hepcidin by adherence and exposure to pro-inflammatory cytokines found in the renal milieu.

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Chronic hypoxia- and cold-induced changes in cardiac enzyme and gene expression in CD-1 mice.

Publication Date: 2010 Aug 26 PMID: 20801191
Authors: Templeman, N. M. - Beaudry, J. L. - Le Moine, C. M. - McClelland, G. B.
Journal: Biochim Biophys Acta

BACKGROUND: In mammals, environmental challenges often result in physical and metabolic cardiac remodeling (i.e. hypertrophy and a shift from lipid to carbohydrate oxidation). While chronic hypoxia and cold are both known to elicit cardiac changes, little is known about their combined effects. METHODS: To investigate the cumulated effects of these two stressors on cardiac physiology, CD-1 mice were exposed for four weeks to normoxia/normothermia, hypoxia, cold, or combined hypoxic-cold. We assessed physical characteristics, left ventricular activities of fatty acid catabolic enzymes short-chain ss-hydroxyacyl-CoA dehydrogenase (SCHAD) and medium-chain acyl-CoA dehydrogenase, and mRNA levels of Acadm, muscle- and liver-type carnitine palmitoyltransferase (Cpt-1beta, Cpt-1alpha), and the transcriptional regulator PPARalpha. RESULTS: 1) Chronic hypoxia reduced SCHAD activity without physical remodeling or mRNA changes; 2) chronic cold lead to reduced SCHAD activity in hypertrophied left ventricles and lowered right ventricular Cpt-1alpha mRNA (compared to chronic hypoxia); and 3) despite causing hypertrophy of both ventricles, chronic exposure to combined hypoxic-cold did not induce significant metabolic remodeling. CONCLUSIONS: In response to environmental challenges, cardiac muscles: 1) show distinct physical and metabolic remodeling; 2) respond to two stressors simultaneously but not additively; and 3) maintain an adult metabolic phenotype with long-term exposure to environmentally realistic hypoxic-cold.

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The Sec translocase.

Publication Date: 2010 Aug 26 PMID: 20801097
Authors: du Plessis, D. J. - Nouwen, N. - Driessen, A. J.
Journal: Biochim Biophys Acta

The vast majority of protein trafficking across or into the bacterial cytoplasmic membrane occurs via the translocon. The translocon consists of the SecYEG complex that forms an evolutionarily conserved heterotrimeric protein conducting membrane channel that functions in conjunction with a variety of ancillary proteins. For post-translational protein translocation, the translocon interacts with the cytosolic motor protein SecA that drives the ATP-dependent stepwise translocation of unfolded polypeptides across the membrane. For the co-translational integration of membrane proteins, the translocon interacts with ribosome-nascent chain complexes and membrane insertion is coupled to polypeptide chain elongation at the ribosome. These process are assisted by YidC and the SecDF(yajC) complex that transiently interact with the translocon. This review summarizes our current understanding of the structure-function relationship of the translocon and its interactions with ancillary components during protein translocation and membrane protein insertion.

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Effects of peptide hydrophobicity on its incorporation in phospholipid membranes - an NMR and ellipsometry study.

Publication Date: 2010 Aug 26 PMID: 20801096
Authors: Oradd, G. - Schmidtchen, A. - Malmsten, M.
Journal: Biochim Biophys Acta

Effects of peptide hydrophobicity on lipid membrane binding, incorporation, and defect formation was investigated for variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQHARASHLGLAR), in anionic 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes. Using a method combination of, e.g., ellipsometry, CD, and fluorescence spectroscopy, it was shown that peptide adsorption, as well as peptide-induced liposome leakage and bactericidal potency against Escherichia coli and Pseudomonas aeruginosa, was promoted by increasing the hydrophobicity of CNY21 through either substituting the two histidines (H) in CNY21 with more hydrophobic leucine (L) residues, or end-tagging with tritryptophan (WWW). Fluorescence spectroscopy revealed that both CNY21WWW and the WWW tripeptide localized to the polar headgroup region of these phospholipid membranes. Deuterium NMR experiments on macroscopically oriented membranes containing fully (palmitoyl) deuterated POPC (POPC-d(31)) demonstrated that both CNY21L and CNY21WWW induced disordering of the lipid membrane. In contrast, for cholesterol-supplemented POPC-d(31) bilayers, peptide-induced disordering was less pronounced in the case of CNY21L, indicating that the peptide is unable to partition to the interior of the lipid membrane in the presence of cholesterol. CNY21WWW, on the other hand, retained its membrane-disordering effect also for cholesterol-supplemented POPC-d(31). These findings were supported by pulsed field gradient NMR experiments where the lateral lipid diffusion was determined in the absence and presence of peptides. Overall, the results provide some mechanistic understanding to previously observed effects of peptide hydrophobization through point mutations and end-tagging, particularly so for complement-based antimicrobial peptides.

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Special issue on estrogen actions in the brain.

Publication Date: 2010 Oct PMID: 20800763
Authors: Casadesus, G.
Journal: Biochim Biophys Acta

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Linker histone subtypes are not generalized gene repressors.

Publication Date: 2010 Aug 25 PMID: 20800709
Authors: Trollope, A. F. - Sapojnikova, N. - Thorne, A. W. - Crane-Robinson, C. - Myers, F. A.
Journal: Biochim Biophys Acta

Antibodies to the six chicken histone H1 subtypes and the variant histone H5 have been used in immunoprecipitations of crosslinked chromatin fragments (xChIPs) to map linker histones across the beta-globin locus and the widely expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and carbonic anhydrase (CA) genes in three cell types: 15-day embryo chicken erythrocytes, 15-day embryo chicken brain and the early erythroid cell line HD24. In erythrocytes, where the beta-adult and beta-hatching genes are active, the H1.01, H1.11L and H1.11R subtypes are substantially depleted throughout the beta-globin locus and the neighboring heterochromatin, in contrast to the other four subtypes, in particular the more abundant H5. Active genes therefore carry high levels of some but not all linker histone subtypes. The situation is similar in HD24 cells, except that substantial depletions are found at the promoters of the adult beta(A) and embryonic beta(rho) and beta(epsilon) genes, despite these genes not yet being active in HD24 cells. The distributions in the brain tissue are characterised by the absence of H1.02, H1.03 and H5 from the hypersensitive site HS3 and from the beta-adult 3' enhancer for the H1.11L and H1.11R subtypes. The data show that although linker histone subtypes play distinct cell-type specific roles in gene regulation, their widespread distribution indicates they are not intrinsically inhibitory to basic chromatin transactions.

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Structure characterization of the 26S proteasome.

Publication Date: 2010 Aug 25 PMID: 20800708
Authors: Kim, H. M. - Yu, Y. - Cheng, Y.
Journal: Biochim Biophys Acta

In all eukaryotic cells, 26S proteasome plays an essential role in the process of ATP-dependent protein degradation. In this review, we focus on structure characterization of the 26S proteasome. Although the progress towards a high-resolution structure of the 26S proteasome has been slow, the recently solved structures of various proteasomal subcomplexes have greatly enhanced our understanding of this large machinery. In addition to having an ATP-dependent proteolytic function, the 26S proteasome is also involved in many non-proteolytic cellular activities, which are often mediated by subunits in its 19S regulatory complex. Thus, we include a detailed discussion of the structures of 19S subunits, including proteasomal ATPases, ubiquitin receptors, deubiquitinating enzymes and subunits that contain PCI domain.

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Distinct regulatory mechanisms of eukaryotic transcriptional activation by SAGA and TFIID.

Publication Date: 2010 Aug 25 PMID: 20800707
Authors: Bhaumik, S. R.
Journal: Biochim Biophys Acta

A growing number of human diseases are linked to abnormal gene expression which is largely controlled at the level of transcriptional initiation. The gene-specific activator promotes the initiation of transcription through its interaction with one or more components of the transcriptional initiation machinery, hence leading to stimulated transcriptional initiation or activation. However, all activator proteins do not target the same component(s) of the transcriptional initiation machinery. Rather, they can have different target specificities, and thus, can lead to distinct mechanisms of transcriptional activation. Two such distinct mechanisms of transcriptional activation in yeast are mediated by the SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (Transcription factor IID) complexes, and are termed as "SAGA-dependent" and "TFIID-dependent" transcriptional activation, respectively. SAGA is the target of the activator in case of SAGA-dependent transcriptional activation, while the targeting of TFIID by the activator leads to TFIID-dependent transcriptional activation. Both the SAGA and TFIID complexes are highly conserved from yeast to human, and play crucial roles in gene activation among eukaryotes. The regulatory mechanisms of eukaryotic transcriptional activation by SAGA and TFIID are discussed here.

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Modulation of Lipid Droplet Size and Lipid Droplet Proteins by Trans-10,cis-12 Conjugated Linoleic Acid Parallels Improvements in Hepatic Steatosis in Obese, Insulin-resistant Rats.

Publication Date: 2010 Aug 25 PMID: 20800698
Authors: Stringer, D. M. - Zahradka, P. - Declercq, V. C. - Ryz, N. R. - Diakiw, R. - Burr, L. L. - Xie, X. - Taylor, C. G.
Journal: Biochim Biophys Acta

The isomer-specific effects of conjugated linoleic acid (CLA) on hepatic steatosis were assessed in fa/fa Zucker rats, a model for insulin resistance and the metabolic syndrome. Eight weeks of feeding trans-10,cis-12 CLA significantly improved glucose tolerance without changing body weight or visceral adipose mass. The trans-10,cis-12 isomer was also associated with reduced liver lipid content, improved liver function and reduced inflammation; these effects were not observed in rats fed the cis-9,trans-11 CLA isomer. Reduced liver lipid content did not correlate with activation of AMP-activated protein kinase or suppressed activation of sterol-regulatory element binding protein-1, two key regulators of hepatic lipid metabolism. Interestingly, rats fed cis-9,trans-11 CLA had fewer cytoplasmic lipid droplets in hepatocytes compared to rats fed control diet, but these droplets were larger in size. Conversely, fa/fa rats fed the trans-10,cis-12 CLA isomer had greater numbers of hepatic lipid droplets that were smaller in size, resulting in overall lower total lipid within these droplets. Changes in lipid droplets were associated with lower hepatic levels of PERILIPIN-2 (formerly known as adipophilin) in rats fed trans-10,cis-12 CLA, whereas amounts of other members of the PERILIPIN family of lipid droplet proteins were unaffected by dietary CLA. However, CLA isomers differentially affected the subcellular localization of these proteins. Treatment of H4IIE rat hepatoma cells with CLA isomers neither prevented or reversed, but rather induced cytoplasmic lipid droplet formation, suggesting that the anti-steatotic effects of trans-10,cis-12 CLA are likely indirect and potentially mediated via increased lipid utilization by peripheral tissues.

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Etoposide induces apoptosis and upregulation of TACE/ADAM17 and ADAM10 in an in vitro male germ cell line model.

Publication Date: 2010 Aug 25 PMID: 20800622
Authors: Lizama, C. - Ludwig, A. - Moreno, R. D.
Journal: Biochim Biophys Acta

Etoposide is a widely used anticancer drug in the treatment of different tumors. Etoposide is known to activate a wide range of intracellular signals, which may in turn induce cellular responses other than apoptosis. ADAM10 and TACE/ADAM17 belong to a family of transmembrane extracellular metalloproteinases involved in paracrine/juxtacrine regulation of many signaling pathways. The aim of this work was to evaluate if etoposide induces upregulation of ADAM10 or TACE/ADAM17 in two cell lines (GC-1 and GC-2) derived from male germ cells. Results showed that etoposide induced apoptosis in a dose-response manner in both GC-1 and GC-2 cells. Apoptosis started to increase 6 hrs after etoposide addition in GC-2 cells, whereas the same was observed 18 hrs after addition to the GC-1 cells. Protein and mRNA levels of ADAM10 and TACE/ADAM17 increased 18 hrs after etoposide was removed from the GC-1 cells. In GC-2 cells, the protein levels of both proteins increased 12 hrs after etoposide was removed. ADAM10 mRNA increased after 3 hrs and then steadily decreased up to 12 hrs after removal, whereas TACE/ADAM17 mRNA decreased after etoposide removal. Finally, apoptosis was prevented in GC-1 and GC-2 cells by the addition of pharmacological inhibitors of ADAM10 and TACE/ADAM17 to the culture medium of etoposide-treated cells. Our results show for the first time that etoposide upregulates ADAM10 and TACE/ADAM17 mRNA and protein levels. In addition, we also show that ADAM10 and TACE/ADAM17 have a role in etoposide-induced apoptosis.

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Inserting membrane proteins: The YidC/Oxa1/Alb3 machinery in bacteria, mitochondria, and chloroplasts.

Publication Date: 2010 Aug 25 PMID: 20800571
Authors: Wang, P. - Dalbey, R. E.
Journal: Biochim Biophys Acta

The evolutionarily conserved YidC/Oxa1p/Alb3 family of proteins plays important roles in the membrane biogenesis in bacteria, mitochondria, and chloroplasts. The members in this family function as novel membrane protein insertases, chaperones, and assembly factors for transmembrane proteins, including energy transduction complexes localized in the bacterial and mitochondrial inner membrane, and in the chloroplast thylakoid membrane. In this review, we will present recent progress with this class of proteins in membrane protein biogenesis and discuss the structure/function relationships.

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Quantitative evaluation of the intrinsic uncoupling modulated by ADP and P(i) in the reconstituted ATP synthase of Escherichia coli.

Publication Date: 2010 Aug 25 PMID: 20800570
Authors: D'Alessandro, M. - Turina, P. - Melandri, B. A.
Journal: Biochim Biophys Acta

The ATP synthase from Escherichia coli was isolated and reconstituted into liposomes. The ATP hydrolysis by these proteoliposomes was coupled to proton pumping, and the ensuing inner volume acidification was measured by the fluorescent probe 9-amino-6-chloro-2-methoxyacridine (ACMA). The ACMA response was calibrated by acid-base transitions, and converted into internal pH values. The rates of internal acidification and of ATP hydrolysis were measured in parallel, as a function of P(i) or ADP concentration. Increasing P(i) monotonically inhibited the hydrolysis rate with a half-maximal effect at 510 muM, whereas it stimulated the acidification rate up to 100-200 muM, inhibiting it only at higher concentrations. The ADP concentration in the assay, due both to contaminant ADP in ATP and to the hydrolysis reaction, was progressively decreased by means of increasing pyruvate kinase activities. Decreasing ADP stimulated the hydrolysis rate, whereas it inhibited the internal acidification rate. The quantitative analysis showed that the relative number of translocated protons per hydrolyzed ATP, i.e. the relative coupling ratio, depended on the concentrations of P(i) and ADP with apparent Kd values of 220 muM and 27 nM respectively. At the smallest ADP concentrations reached, and in the absence of P(i), the coupling ratio dropped down to 15% relative to the value observed at the highest ADP and P(i) concentrations tested. In addition, the data indicate the presence of two ADP and P(i) binding sites, of which only the highest affinity one is related to changes in the coupling ratio.

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Ubiquinol (QH(2)) functions as a negative regulator of purine nucleotide-inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein.

Publication Date: 2010 Aug 25 PMID: 20800569
Authors: Woyda-Ploszczyca, A. - Jarmuszkiewicz, W.
Journal: Biochim Biophys Acta

We compared the influence of different adenine and guanine nucleotides on the free fatty acid-induced uncoupling protein (UCP) activity in non-phosphorylating A. castellanii mitochondria when the membranous ubiquinone (Q) redox state was varied. The purine nucleotides exhibit an inhibitory effect in the following descending order: GTP>ATP>GDP>ADP>>GMP>AMP. The efficiency of guanine and adenine nucleotides to inhibit UCP-sustained uncoupling in A. castellanii mitochondria depends on the Q redox state. Inhibition by purine nucleotides can be increased with decreasing Q reduction level (thereby ubiquinol, QH(2) concentration) even with nucleoside monophosphates that are very weak inhibitors at the initial respiration. On the other hand, the inhibition can be alleviated with increasing Q reduction level (thereby QH(2) concentration). The most important finding was that ubiquinol (QH(2)) but not oxidised Q functions as a negative regulator of UCP inhibition by purine nucleotides. For a given concentration of QH(2), the LA-induced GTP-inhibited H(+) leak was the same for two types of A. castellanii mitochondria that differ in the endogenous Q content. When availability of the inhibitor (GTP) or the negative inhibition modulator (QH(2)) was changed, a competitive influence on the UCP activity was observed. QH(2) decreases the affinity of UCP for GTP and, vice versa, GTP decreases the affinity of UCP for QH(2). These results describe the kinetic mechanism of regulation of UCP affinity for purine nucleotides by endogenous QH(2) in the mitochondria of a unicellular eukaryote.

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Mouse models of neurological disorders.

Publication Date: 2010 Oct PMID: 20800183
Authors: Howlett, D.
Journal: Biochim Biophys Acta

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Protein dynamics and ligand migration interplay as studied by computer simulation.

Publication Date: 2010 Aug 23 PMID: 20797453
Authors: Arroyo-Manez, P. - Bikiel, D. E. - Boechi, L. - Capece, L. - Di Lella, S. - Estrin, D. A. - Marti, M. A. - Moreno, D. M. - Nadra, A. D. - Petruk, A. A.
Journal: Biochim Biophys Acta

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases.

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Kinetic crystallography by Raman Microscopy.

Publication Date: 2010 Aug 23 PMID: 20797452
Authors: Carey, P. R. - Chen, Y. - Gong, B. - Kalp, M.
Journal: Biochim Biophys Acta

Raman spectra, obtained using a Raman microscope, offer an unique and incisive approach to follow interactions and reactions inside a single crystal under soak-in or soak-out conditions. The utility of this approach derives from the finding that the Raman spectra from single macromolecular crystals, under normal (non-resonance) conditions, are extremely stable, with a low "light background," and provide ideal platforms for Raman difference spectroscopy. In turn, this allows the interrogation of sub-molecular changes in very large and complex macromolecular environments. There is often great synergy with X-ray crystallography, with the Raman spectroscopist providing crystallography colleagues with the best soak-in conditions to generate a targeted intermediate for flash freezing and X-ray analysis. On the other hand, X-ray structures at points along a reaction pathway provide invaluable benchmarks for interpreting the Raman data from populations seen by Raman to be changing in real-time. These principles will be illustrated by two reactions: The first involves a complex, branching reaction pathway underlying the inhibition of beta-lactamases by clinically important pharmaceutical compounds, where different combinations of drug and enzyme function in different regions of the pathway. The second shows how temporal data can be derived for several events in the initiation step of RNA synthesis-more specifically, when one GTP molecule is joined to one ATP molecule to form a G*A dimer in the active site of a 115,000 Dalton crystalline RNA polymerase. Finally, we will summarize the extension of Raman microscopy to nucleic acid crystals and the information that has been obtained for RNA-based enzymes.

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Whole blood, flow-chamber studies in real-time indicate a biphasic role for thymosin beta-4 in platelet adhesion.

Publication Date: 2010 Aug 23 PMID: 20797426
Authors: Kaur, H. - Heeney, R. - Carriveau, R. - Sosne, G. - Mutus, B.
Journal: Biochim Biophys Acta

BACKGROUND: Thymosin beta 4 (Tbeta(4)) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti inflammatory agent and promotes corneal wound healing. METHODS: In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins-fibrinogen and collagen-were immobilized onto the middle ~25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tbeta(4), on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers. RESULTS: The binding data from the flow chambers, indicated that the both the rate constant of platelet deposition (average: 0.026+/-0.0015s(-1), corresponding to a half-life of 26.7s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tbeta(4) (0.2muM to 0.5muM) increased both the rate constant of platelet deposition by ~1.5-fold (i.e. half-life decreased from 26.7s to 17.6s) and the total number of deposited platelets by ~3-fold. However at higher concentrations (>1muM) the Tbeta(4) -potentiating effect was diminished to near control levels. Tbeta(4) did interact with fibrinogen with an estimated K(D) of~126+/-18 nM or 66+/-20 nM under equilibrium or flow, respectively. CONCLUSION: These results suggest that Tbeta(4) could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tbeta(4) could also bind to fibrinogen and as its concentration increased would prevent platelet-fibrinogen interactions resulting in the attenuation of platelet deposition. GENERAL SIGNIFICANCE: This work suggests that Tbeta(4) might have a dual role in platelet function.

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Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment.

Publication Date: 2010 Aug 23 PMID: 20797386
Authors: Huc-Brandt, S. - Marcellin, D. - Graslin, F. - Averseng, O. - Bellanger, L. - Hivin, P. - Quemeneur, E. - Basquin, C. - Navarro, V. - Pourcher, T. - Darrouzet, E.
Journal: Biochim Biophys Acta

The sodium/iodide symporter (NIS) is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. NIS is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably-expressing various human NIS recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of hNIS was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a NIS C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human NIS protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely-tune iodide accumulation through hNIS regulation.

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Progress in tracing the evolutionary paths of cytochrome P450.

Publication Date: 2010 Aug 21 PMID: 20736090
Authors: Nelson, D. R.
Journal: Biochim Biophys Acta

The state of cytochrome P450 sequence accumulation in different phyla is summarized. 12,456 P450s are currently named, with about 6000 more that are known, but not yet named. As the number of genomes sequenced moves from a few dozen to an inevitable few thousand, issues of nomenclature are discussed. Orthology will be the guiding principle for naming across related genomes such as vertebrates. Even if 1000 vertebrate genomes are sequenced there will still be only 19 CYP families in vertebrates. The variable clusters of genes in families CYP2, CYP3 and CYP4 may pose challenges for naming as 1:1 orthologs do not necessarily exist. The value of synteny across genomes is emphasized as a tool for deep time evolutionary studies of P450s in animals. There is evidence that macrosynteny may be useful in tracing the origin of animal CYP clans. The concept of saturation of sequence space is described and used to estimate how complete our knowledge is of P450s in different phyla. The special niche of filamentous fungal P450s acting in secondary metabolite gene clusters is discussed. From one quarter to one third of P450s in these fungi may be dedicated to these roles.

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Exploring the positional importance of aromatic residues and lysine in the interactions of peptides with the P. falciparum Hsp70-1.

Publication Date: 2010 Aug 21 PMID: 20736089
Authors: Misra, G. - Ramachandran, R.
Journal: Biochim Biophys Acta

P. falciparum harbors an essential relict plastid called the apicoplast that is involved in several important biosynthetic processes. Over 500 nuclear encoded proteins are imported into the organelle that is now recognized as an important therapeutic target. These proteins contain an N-terminal transit peptide sequence essential for apicoplast targeting during which the P. falciparum Hsp70-1 plays an important role. In the present study, we have focused on the in vitro interactions of PfHsp70-1 with synthetic peptides endowed with transit peptide like features. The peptides exhibit higher affinity for PfHsp70-1in the presence of ADP compared to ATP. The results highlight the positional importance of selected residues in the designed peptides for affinity. They suggest that better peptide affinity for the protein requires a Lys at second position, retention of aromatic residue at the last position and absence of acidic residues at any position in the transit peptides. Overall the present work is the first in vitro fluorescence-based study of PfHsp70-1 with peptides possessing transit peptide like features.

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Enzymological studies on the biosynthesis of N-acylethanolamines.

Publication Date: 2010 Aug 21 PMID: 20736084
Authors: Ueda, N. - Tsuboi, K. - Uyama, T.
Journal: Biochim Biophys Acta

Ethanolamides of different long-chain fatty acids constitute a class of endogenous lipid molecules generally called N-acylethanolamines (NAEs). They contain N-arachidonoylethanolamine (anandamide), N-palmitoylethanolamine, and N-oleoylethanolamine, which receive considerable attention because of their actions as an endogenous cannabinoid receptor ligand (endocannabinoid), an anti-inflammatory substance, and an appetite-suppressing substance, respectively. Identification of their biosynthetic routes in animal tissues and molecular characterization of the enzymes involved are essential for better understanding of physiological importance of NAEs as well as development of enzyme inhibitors as possible therapeutic drugs. In the classical "transacylation-phosphodiesterase pathway", NAEs are formed from glycerophospholipids via N-acylphosphatidylethanolamine (NAPE), an unusual derivative of phosphatidylethanolamine with a third acyl chain attached to the amino group, by sequential catalyses by Ca(2+)-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D. However, recent studies reveal that NAE-generating pathways are more complex than presumed before. In this review article, we will focus on recent findings regarding mammalian enzymes that are involved or might be involved in the biosynthesis of NAEs.

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Repercussion of a deficiency in mitochondrial ss-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ss-oxidation cycle in Aspergillus nidulans.

Publication Date: 2010 Aug 21 PMID: 20736083
Authors: Magliano, P. - Sanglard, D. - Poirier, Y.
Journal: Biochim Biophys Acta

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ss-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ss-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ss-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ss-oxidation cycle can handle both long and short-chain intermediates. While the foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ss-oxidation is actually enhanced in its absence. While no change in PHA was observed in the scdAechA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in scdAechA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ss-oxidation cycle when the mitochondrial ss-oxidation pathway is defective.

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The complexity of HDL.

Publication Date: 2010 Aug 21 PMID: 20736082
Authors: Francis, G. A.
Journal: Biochim Biophys Acta

Plasma high density lipoprotein cholesterol (HDL-C) levels are inversely associated with coronary artery disease risk in large epidemiologic studies. This rule, however, has many exceptions in individual patients, and evidence suggests other facets of high density lipoprotein particle biology not captured by measuring HDL-C levels are responsible for HDL's effects in vivo. This article reviews the evidence for the protective nature of HDL, current evidence from animal and human studies regarding HDL-based therapies, the major steps in HDL particle formation and metabolism, alterations leading to dysfunctional HDL in diabetes and inflammatory states, and potential alternatives to HDL-C to measure HDL function and predict its protective value clinically.

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Evidence for the cytotoxic effects of mycobacterium tuberculosis phospholipases C towards macrophages.

Publication Date: 2010 Aug 21 PMID: 20736081
Authors: N'goma, J. C. - Schue, M. - Carriere, F. - Geerlof, A. - Canaan, S.
Journal: Biochim Biophys Acta

Phospholipases C (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterized, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipases C (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterizing biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipases C can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind.

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Mitophagy and Parkinson's disease: The PINK1-parkin link.

Publication Date: 2010 Aug 21 PMID: 20736035
Authors: Deas, E. - Wood, N. W. - Plun-Favreau, H.
Journal: Biochim Biophys Acta

The study of rare, inherited mutations underlying familial forms of Parkinson's disease has provided insight into the molecular mechanisms of disease pathogenesis. Mutations in these genes have been functionally linked to several key molecular pathways implicated in other neurodegenerative disorders, including mitochondrial dysfunction, protein accumulation and the autophagic-lysosomal pathway. In particular, the mitochondrial kinase PINK1 and the cytosolic E3 ubiquitin ligase parkin act in a common pathway to regulate mitochondrial function. In this review we discuss the recent evidence suggesting that the PINK1/parkin pathway also plays a critical role in the autophagic removal of damaged mitochondria-mitophagy.

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Apo A-II participates in HDL-liposome interaction by the formation of new pre-beta mobility particles and the modification of liposomes.

Publication Date: 2010 Aug 21 PMID: 20732453
Authors: Wroblewska, M. - Czyzewska, M. - Wolska, A. - Kortas-Stempak, B. - Szutowicz, A.
Journal: Biochim Biophys Acta

Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-beta HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-beta mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-beta fraction was significantly higher than those accepted by alpha-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-beta mobility fraction. The relative contents of PL and apo A-I in pre-beta fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-beta fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-beta fraction protein mass. Both liposomes and pre-beta fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-beta mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL-liposome interaction may influence the anti-atherogenic properties of liposomes.

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A role for the scavenger receptor, class B type I in high density lipoprotein dependent activation of cellular signaling pathways.

Publication Date: 2010 Aug 21 PMID: 20732452
Authors: Al-Jarallah, A. - Trigatti, B. L.
Journal: Biochim Biophys Acta

High density lipoprotein (HDL) levels are inversely proportional to the risk of coronary heart disease. HDL mediates various anti-atherogenic pathways including reverse cholesterol transport from cells of the arterial wall to the liver and steroidogenic tissues. In addition HDL activates various intracellular signaling events that confer atheroprotection. The HDL receptor, scavenger receptor class B type I (SR-BI) has been implicated directly and indirectly in HDL induced signaling. The aim of this review is to summarize the role of SR-BI in HDL induced signaling in the vasculature.

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Hypoxia-inducible factor 1: Regulator of mitochondrial metabolism and mediator of ischemic preconditioning.

Publication Date: 2010 Aug 21 PMID: 20732359
Authors: Semenza, G. L.
Journal: Biochim Biophys Acta

Hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability by regulating gene expression. A critical cell-autonomous adaptive response to chronic hypoxia controlled by HIF-1 is reduced mitochondrial mass and/or metabolism. Exposure of HIF-1-deficient fibroblasts to chronic hypoxia results in cell death due to excessive levels of reactive oxygen species (ROS). HIF-1 reduces ROS production under hypoxic conditions by multiple mechanisms including: a subunit switch in cytochrome c oxidase from the COX4-1 to COX4-2 regulatory subunit that increases the efficiency of complex IV; induction of pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; induction of BNIP3, which triggers mitochondrial selective autophagy; and induction of microRNA-210, which blocks assembly of Fe/S clusters that are required for oxidative phosphorylation. HIF-1 is also required for ischemic preconditioning and this effect may be due in part to its induction of CD73, the enzyme that produces adenosine. HIF-1-dependent regulation of mitochondrial metabolism may also contribute to the protective effects of ischemic preconditioning.

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Sigma-1 receptors amplify dopamine D1 receptor signaling at presynaptic sites in the prelimbic cortex.

Publication Date: 2010 Aug 20 PMID: 20732358
Authors: Fu, Y. - Zhao, Y. - Luan, W. - Dong, L. Y. - Dong, Y. - Lai, B. - Zhu, Y. - Zheng, P.
Journal: Biochim Biophys Acta

Sigma-1 receptors are highly expressed in the brain. The downstream signaling mechanisms associated with the sigma-1 receptor activation have been shown to involve the activation of protein kinase C (PKC), the control of Ca(2) homoeostasis and the regulation of voltage- and ligand-gated ion channels. But few studies examined the regulatory effect of sigma-1 receptors on metabotropic receptor signaling. The present paper studied the regulatory effect of sigma-1 receptors on the signaling of dopamine D1 receptors, one of metabotropic receptors, by examining the effect of sigma-1 receptor agonists on the D1 receptor agonist-induced cAMP-dependent protein kinase (PKA) activation at presynaptic sites using the synaptosomes from the prelimbic cortex. The results showed that sigma-1 receptor agonists alone had no effects on the PKA activity, but could amplify the D1 receptor agonist-induced PKA activation. The sigma-1 receptor agonist also amplified the membrane-permeable analog of cAMP- and the adenylyl cyclase (AC) activator-induced PKA activation, but did not on the D1 receptor agonist-induced AC activation. The conventional PKC (cPKC), especially the PKCbetaI, and the extracellular Ca(2+) influx through L-type Ca(2+) channels might play key roles in the amplifying effect of the sigma-1 receptor agonists. The activation of PKC by sigma-1 receptor agonists was the upstream event of the increase in the intrasynaptosomal Ca(2+) concentration. These results suggest that sigma-1 receptors may amplify the D1 receptor agonist-induced PKA activation by sigma-1 receptors - cPKC (especially the PKCbetaI) - L-type Ca(2+) channels - Ca(2+) - AC and/or cAMP signaling pathway.

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Learning from oncocytic tumors: Why choose inefficient mitochondria?

Publication Date: 2010 Aug 21 PMID: 20732299
Authors: Gasparre, G. - Romeo, G. - Rugolo, M. - Porcelli, A. M.
Journal: Biochim Biophys Acta

A prominent role for mitochondrial genes and metabolism has been recently characterized in oncocytic transformation of cancer cells. From mitochondrial ultrastructure alterations to respiratory complexes disruption and mutations within mitochondrial genes, oncocytic tumors present with a plethora of features that have helped understand the role that these organelles and their fundamental metabolic functions may play in cancer development. The history of this under-diagnosed subset of tumors and the bioenergetic implications of their mitochondrial derangement are discussed in this review along with the opportunities that oncocytic tumors offer to draw general conclusions on the involvement of mitochondria in cancer.

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The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides.

Publication Date: 2010 Aug 21 PMID: 20732298
Authors: Huxley, L. - Quirk, P. G. - Cotton, N. P. - White, S. A. - Jackson, J. B.
Journal: Biochim Biophys Acta

In its forward direction, transhydrogenase couples the reduction of NADP(+) by NADH to the outward translocation of protons across the membrane of bacteria and animal mitochondria. The enzyme has three components: dI and dIII protrude from the membrane and dII spans the membrane. Hydride transfer takes place between nucleotides bound to dI and dIII. Studies on the kinetics of a lag phase at the onset of a "cyclic reaction" catalysed by complexes of the dI and dIII components of transhydrogenase from Rhodospirillum rubrum, and on the kinetics of fluorescence changes associated with nucleotide binding, reveal two features. Firstly, the binding of NADP(+) and NADPH to dIII is extremely slow, and is probably limited by the conversion of the occluded to the open state of the complex. Secondly, dIII can also bind NAD(+) and NADH. Extrapolating to the intact enzyme this binding to the "wrong" site could lead to slip: proton translocation without change in the nucleotide redox state, which would have important consequences for bacterial and mitochondrial metabolism.

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Approaches for targeting mitochondria in cancer therapy.

Publication Date: 2010 Aug 20 PMID: 20732297
Authors: D'Souza, G. G. - Wagle, M. A. - Saxena, V. - Shah, A.
Journal: Biochim Biophys Acta

The recognition of the role that mitochondria play in human health and disease is evidenced by the emergence in recent decades of a whole new field of "Mitochondrial Medicine". Molecules located on or inside mitochondria are considered prime pharmacological targets and a wide range of efforts are under way to exploit these targets to develop targeted therapies for various diseases including cancer. However the concept of targeting, while seemingly simple in theory, has multiple subtly different practical approaches. The focus of this article is to highlight these differences in the context of a discussion on the current status of various mitochondria-targeted approaches to cancer therapy.

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Bioactive food components, cancer cell growth limitation and reversal of glycolytic metabolism.

Publication Date: 2010 Aug 21 PMID: 20732296
Authors: Keijer, J. - Bekkenkamp-Grovenstein, M. - Venema, D. - Dommels, Y. E.
Journal: Biochim Biophys Acta

Cancer cells are resistant to apoptosis and show a shift in energy production from mitochondrial oxidative phosphorylation to cytosolic glycolysis. Apoptosis resistance and metabolic reprogramming are linked in many cancer cells and both processes center on mitochondria. Clearly, mutated cancer cells escape surveillance and turn into selfish cells. However, many of the mechanisms that operate cellular metabolic control still function in cancer cells. This review describes the metabolic importance of glucose and glutamine, glycolytic enzymes, oxygen, growth cofactors and mitochondria and focuses on the potential role of bioactive food components, including micronutrients. The role of B- and A-vitamin cofactors in (mitochondrial) metabolism is highlighted and the cancer protective potential of omega-3 fatty acids and several polyphenols is discussed in relation to metabolic reprogramming, including the mechanisms that may be involved. Furthermore, it is shown that cancer cell growth reduction by limiting the growth cofactor folic acid seems to be associated with reversal of metabolic reprogramming. Altogether, reversal of metabolic reprogramming may be an attractive strategy to increase susceptibility to apoptotic surveillance. Food bioactive components that affect various aspects of metabolism may be important tools to reverse glycolytic to oxidative metabolism and enhance sensitivity to apoptosis. The success of such a strategy may depend on several actors, acting in concert. Growth cofactors may be one of these, which call for careful (re)evaluation of their function in normal and in cancer metabolism.

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Maternally inherited susceptibility to cancer.

Publication Date: 2010 Aug 21 PMID: 20732295
Authors: Bayona-Bafaluy, M. P. - Lopez-Gallardo, E. - Montoya, J. - Ruiz-Pesini, E.
Journal: Biochim Biophys Acta

Tumor microenvironment promotes mtDNA mutations. A number of these mutations will affect cell metabolism and increase cell survival. These mutations are positively selected and contribute to other tumor features, such as extracellular matrix remodeling and angiogenic processes, thus favoring metastases. Like somatic mutations, although with less marked effects, some mtDNA population polymorphisms will affect OXPHOS function, cell metabolism, and homeostasis. Thus, they could behave as inherited susceptibility factors for cancer. However, in addition to epidemiological evidence, other more direct clues are required. The cybrid approach can help to clarify this issue.

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Emerging roles of the 26S proteasome in nuclear hormone receptor-regulated transcription.

Publication Date: 2010 Aug 20 PMID: 20728592
Authors: Keppler, B. R. - Archer, T. K. - Kinyamu, H. K.
Journal: Biochim Biophys Acta

The mechanisms by which nuclear hormone receptors (NHRs) regulate transcription are highly dynamic and require interplay between a myriad of regulatory protein complexes including the 26S proteasome. Protein degradation is the most well-established role of the proteasome; however, an increasing body of evidence suggests that the 26S proteasome may regulate transcription in proteolytic and nonproteolytic mechanisms. Here we review how these mechanisms may apply to NHR-mediated transcriptional regulation.

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Dynamics of reversible protein phosphorylation in thylakoids of flowering plants: The roles of STN7, STN8 and TAP38.

Publication Date: 2010 Aug 20 PMID: 20728426
Authors: Pesaresi, P. - Pribil, M. - Wunder, T. - Leister, D.
Journal: Biochim Biophys Acta

Phosphorylation is the most common post-translational modification found in thylakoid membrane proteins of flowering plants, targeting more than two dozen subunits of all multiprotein complexes, including some light-harvesting proteins. Recent progress in mass spectrometry-based technologies has led to the detection of novel low-abundance thylakoid phosphoproteins and localised their phosphorylation sites. Three of the enzymes involved in phosphorylation/dephosphorylation cycles in thylakoids, the protein kinases STN7 and STN8 and the phosphatase TAP38/PPH1, have been characterised in the model species Arabidopsis thaliana. Differential protein phosphorylation is associated with changes in illumination and various other environmental parameters, and has been implicated in several acclimation responses, the molecular mechanisms of which are only partly understood. The phenomenon of State Transitions, which enables rapid adaptation to short-term changes in illumination, has recently been shown to depend on reversible phosphorylation of LHCII by STN7-TAP38/PPH1. STN7 is also necessary for long-term acclimation responses that counteract imbalances in energy distribution between PSII and PSI by changing the rates of accumulation of their reaction-centre and light-harvesting proteins. Another aspect of photosynthetic acclimation, the modulation of thylakoid ultrastructure, depends on phosphorylation of PSII core proteins, mainly executed by STN8. Here we review recent advances in the characterisation of STN7, STN8 and TAP38/PPH1, and discuss their physiological significance within the overall network of thylakoid protein phosphorylation.

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Epidermal growth factor regulates PAI-1 expression via activation of the transcription factor Elk-1.

Publication Date: 2010 Aug 19 PMID: 20727996
Authors: Wyrzykowska, P. - Stalinska, K. - Wawro, M. - Kochan, J. - Kasza, A.
Journal: Biochim Biophys Acta

PAI-1 (plasminogen activator inhibitor-1) in breast cancer cells is involved in tumour development and metastasis of breast cancer cells. PAI-1 function and the regulation of its expression have been precisely investigated. Here we report that EGF, which promotes breast cancer tumour growth and survival, rapidly induces PAI-1 expression in the breast adenocarcinoma cell line MCF-7 through the activation of the transcription factor Elk-1. We have found that the PAI-1 promoter fragment (-140 to +173) containing the Ets consensus binding site is activated by Elk-1. Chromatin immunoprecipitation analysis confirms in vivo binding of Elk-1 to the PAI-1 promoter and demonstrates that Elk-1 phosphorylation on the Ets binding site is EGF-dependent.

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Five monomeric hemocyanin subunits from Portunus trituberculatus: Purification, spectroscopic characterization, and quantitative evaluation of phenol monooxygenase activity.

Publication Date: 2010 Aug 18 PMID: 20727990
Authors: Fujieda, N. - Yakiyama, A. - Itoh, S.
Journal: Biochim Biophys Acta

Five kinds of monomeric subunits of arthropod hemocyanin have been isolated from swimming crab Portunus trituberculatus hemolymph. The copper centers holding a peroxo species, [(mu-eta(2):eta(2)-peroxo)dicopper(II)], of these subunits exhibited almost the same UV-vis and visible region CD spectroscopic properties, indicating that they have a similar copper coordination geometry and an electronic structure. Under anaerobic conditions, the oxy-forms of the monomeric subunits were stable in 0.5M borate buffer (pH 9.0) and reacted with 4-methylphenol (p-cresol) to show the phenolases (cresolase/phenol monooxygenase) activity in the presence of urea. To compare the phenolase (monooxygenase) reactivity, the reactivity of the isolated subunits has been examined quantitatively by using a simplified catalytic system, where the initial product catechol is trapped with borate anion of the buffer solution to prevent following catecholase reaction (Yamazaki and Itoh, 2003). The far-UV region CD spectra were measured in order to clarify the relationship between the content of the secondary structure and the phenolase reactivity. Even though the monomeric subunits exhibit a weak catalytic phenol monooxygenase activity, addition of urea (3M) significantly enhances their catalytic activity. The differences of the phenolase activity among the monomeric subunits has been discussed on the basis of the spectroscopic analysis and reactivity studies in order to shed light on the enzymatic function of the arthropod hemocyanin in vivo.

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Surface localized and extracellular Glyceraldehyde-3-phosphate dehydrogenase of Bacillus anthracis is a plasminogen binding protein.

Publication Date: 2010 Aug 18 PMID: 20727989
Authors: Matta, S. K. - Agarwal, S. - Bhatnagar, R.
Journal: Biochim Biophys Acta

The role of anchorless proteins on the surface of most pathogenic microorganisms has long been studied in context to their interactions with multiple host proteins, facilitating the dissemination of pathogen within the host tissues. In order to gain more insights into anthrax pathogenesis, we hereby report the presence of a prominent moonlighting enzyme, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface and in the extracellular medium of Bacillus anthracis. Out of the three heterologously expressed recombinant isoforms, rGapA (with 334 amino acids) showed a significant NAD(+) mediated GAPDH activity, whereas rGapB (with 342 amino acids) showed a slight activity with NADP(+). The rGapN (with 479 amino acids) was enzymatically inactive with either NAD(+) or NADP(+). GapA was ascertained to be present in the extracellular medium and on the surface of B. anthracis. On the other hand, GapN was absent from both the surface and extracellular medium, whereas GapB was scarcely present on the surface of B. anthracis. Human plasminogen predominantly interacted with the rGapA isoform at physiological concentrations and the interaction was found to be lysine dependent. Immunization with rGapA resulted in a significant protection upon challenge with Bacillus anthracis in the murine model.

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Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae.

Publication Date: 2010 Aug 19 PMID: 20727985
Authors: Rajakumari, S. - Rajasekharan, R. - Daum, G.
Journal: Biochim Biophys Acta

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction [K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using [(3)H]inositol, [(32)P]orthophosphate, [(3)H]palmitate and [(14)C]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids.

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Secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis C virus by NMR spectroscopy.

Publication Date: 2010 Aug 18 PMID: 20727850
Authors: Cook, G. A. - Opella, S. J.
Journal: Biochim Biophys Acta

P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle 'q-titration,' demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.

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Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.

Publication Date: 2010 Aug 19 PMID: 20727849
Authors: Zhang, L. - Aleksandrov, L. A. - Riordan, J. R. - Ford, R. C.
Journal: Biochim Biophys Acta

The domain organisation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was studied using electron microscopy of detergent-solubilised dimeric complexes. Ni-NTA nanogold labelling data suggest that in the nonphosphorylated, nucleotide-free state, the C-terminus is intimately associated with the cytoplasmic ATP-binding regions, whilst part of the regulatory domain occupies a position close to the cytoplasmic surface of the lipid membrane. Removal of the entire second nucleotide binding domain (NBD2) results in a deficit in the CFTR structure that is consistent with the size and shape of a single NBD. The data suggest that NBD2 lies closer to the C2 symmetry axis than the first nucleotide binding domain (NBD1) and that NBD2 from one CFTR monomer also contacts NBD1 from the opposing one. These data suggest that current homology models for CFTR based on other ATP-binding cassette proteins appear to be reasonable, at least to low resolution. We also find that Ni-NTA nanogold labelling of an internal hexa-Histidine sequence is a valuable approach to locate individual protein domains.

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Comparative NMR studies demonstrate profound differences between two viroporins: p7 of HCV and Vpu of HIV-1.

Publication Date: 2010 Aug 18 PMID: 20727848
Authors: Cook, G. A. - Zhang, H. - Park, S. H. - Wang, Y. - Opella, S. J.
Journal: Biochim Biophys Acta

The p7 protein from hepatitis C virus and the Vpu protein from HIV-1 are members of the viroporin family of small viral membrane proteins. It is essential to determine their structures in order to obtain an understanding of their molecular mechanisms and to develop new classes of anti-viral drugs. Because they are membrane proteins, it is challenging to study them in their native phospholipid bilayer environments by most experimental methods. Here we describe applications of NMR spectroscopy to both p7 and Vpu. Isotopically labeled p7 and Vpu samples were prepared by heterologous expression in bacteria, initial isolation as fusion proteins, and final purification by chromatography. The purified proteins were studied in the model membrane environments of micelles by solution NMR spectroscopy and in aligned phospholipid bilayers by solid-state NMR spectroscopy. The resulting structural findings enable comparisons to be made between the two proteins, demonstrating that they have quite different architectures. Most notably, Vpu has one trans-membrane helix and p7 has two trans-membrane helices; in addition, there are significant differences in the structures and dynamics of their internal loop and terminal regions.

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Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

Publication Date: 2010 Aug 17 PMID: 20727375
Authors: Conte, L. - Trumpower, B. L. - Zara, V.
Journal: Biochim Biophys Acta

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homo-dimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two super-complexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the super-complexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly.

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Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium perfringens alpha-toxin.

Publication Date: 2010 Aug 17 PMID: 20727345
Authors: Urbina, P. - Flores-Diaz, M. - Alape-Giron, A. - Alonso, A. - Goni, F. M.
Journal: Biochim Biophys Acta

alpha-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the alpha-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that alpha-toxin lipase activities have distinct, but partially overlapping and interacting active sites.

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The proteasome and its regulatory roles in gene expression.

Publication Date: 2010 Aug 17 PMID: 20723625
Authors: Kwak, J. - Workman, J. L. - Lee, D.
Journal: Biochim Biophys Acta

Cumulative evidence indicates that the proteasome, which is mainly known as a protein-degrading machine, is very essential for gene expression. Destructive functions of the proteasome, i.e., ubiquitin-dependent proteolytic activity, are significant for activator localization, activator destruction, co-activator/repressor destruction and PIC disassembly. Non-proteolytic functions of the proteasome are important for recruitment of activators and co-activators to promoters, ubiquitin-dependent histone modification, transcription elongation and possibly maturation of mRNA via the facilitation of mRNA export from the nucleus to the cytoplasm. In this review, we discuss how the proteasome regulates transcription at numerous stages during gene expression.

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Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors eIF4F, eIF4B and PABP.

Publication Date: 2010 Aug 17 PMID: 20723624
Authors: Yumak, H. - Khan, M. A. - Goss, D. J.
Journal: Biochim Biophys Acta

We have investigated the effects of poly(A)-tail on binding of eIF4F, eIF4B and PABP with tobacco etch virus (TEV) IRES RNA. The fluorescence anisotropy data showed that the addition of poly(A)(20) increases the binding affinity of eIF4F.4B and eIF4F.PABP complexes to IRES RNA ~2- and 4-fold, respectively. However, the binding affinity of eIF4F with PK1 was enhanced ~11-fold with the addition of PABP, eIF4B, and poly(A)(20) together. Whereas, poly(A)(20) alone increases the binding affinity of eIF4F.4B.PABP with PK1 RNA about 3-fold, showing an additive effect rather than the large increase in affinity as shown for cap binding. Thermodynamic data showed that PK1 RNA binding to protein complexes in the presence of poly(A)(20) was enthalpy-driven and entropy-favorable. Poly(A)(20) decreased the entropic contribution 75% for binding of PK1 RNA to eIF4F.4B.PABP as compared to eIF4F alone, suggesting reduced hydrophobic interactions for complex formation and an overall conformational change. Overall, these results demonstrate the first direct effect of poly(A) on the equilibrium and thermodynamics of eIF4F and eIF4F.4B.PABP with IRES-RNA.

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Strategies to inhibit viral protein nuclear import: HIV-1 as a target.

Publication Date: 2010 Aug 16 PMID: 20719241
Authors: Levin, A. - Loyter, A. - Bukrinsky, M.
Journal: Biochim Biophys Acta

Nuclear import is a critical step in the life cycle of HIV-1. During the early (preintegration) stages of infection, HIV-1 has to transport its preintegration complex into the nucleus for integration into the host cell chromatin, while at the later (postintegration) stages viral regulatory proteins Tat and Rev need to get into the nucleus to stimulate transcription and regulate splicing and nuclear export of subgenomic and genomic RNAs. Given such important role of nuclear import in HIV-1 life cycle, this step presents an attractive target for antiviral therapeutic intervention. In this review, we describe the current state of our understanding of the interactions regulating nuclear import of the HIV-1 preintegration complex and describe current approaches to inhibit it.

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Probing membrane topology of the antimicrobial peptide distinctin by solid-state NMR spectroscopy in zwitterionic and charged lipid bilayers.

Publication Date: 2010 Aug 16 PMID: 20719234
Authors: Verardi, R. - Traaseth, N. J. - Shi, L. - Porcelli, F. - Monfregola, L. - De Luca, S. - Amodeo, P. - Veglia, G. - Scaloni, A.
Journal: Biochim Biophys Acta

Distinctin is a 47-residue antimicrobial peptide, which interacts with negatively charged membranes and is active against Gram-positive and Gram-negative bacteria. Its primary sequence comprises two linear chains of 22 (chain 1) and 25 (chain 2) residues, linked by a disulfide bridge between Cys19 of chain 1 and Cys23 of chain 2. Unlike other antimicrobial peptides, distinctin in the absence of the lipid membrane has a well-defined three-dimensional structure, which protects it from protease degradation. Here, we used static solid-state NMR spectroscopy to study the topology of distinctin in lipid bilayers. We found that, in mechanically aligned lipid bilayers (charged or zwitterionic), this heterodimeric peptide adopts an ordered conformation absorbed on the surface of the membrane, with the long helix (chain 2), approximately parallel to the lipid bilayer (~5 degrees from the membrane plane) and the short helix (chain 1) forming a ~24 degrees angle. Since at lipid-to-protein molar ratio of 50:1, the peptide does not disrupt the macroscopic alignment of either charged or zwitterionic lipid bilayers, it is possible that higher concentrations might be needed for the hypothesized pore formation, or alternatively, distinctin elicits its cell disruption action by other mechanisms.

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NAD: A master regulator of transcription.

Publication Date: 2010 Aug 13 PMID: 20713194
Authors: Ghosh, S. - George, S. - Roy, U. - Ramachandran, D. - Kolthur-Seetharam, U.
Journal: Biochim Biophys Acta

Cellular processes such as proliferation, differentiation and death are intrinsically dependent upon the redox status of a cell. Among other indicators of redox flux, cellular NAD(H) levels play a predominant role in transcriptional reprogramming. In addition to this, normal physiological functions of a cell are regulated in response to perturbations in NAD(H) levels (for example, due to alterations in diet/metabolism) to maintain homeostatic conditions. Cells achieve this homeostasis by reprogramming various components that include changes in chromatin structure and function (transcription). The interdependence of changes in gene expression and NAD(H) is evolutionarily conserved and is considered crucial for the survival of a species (by affecting reproductive capacity and longevity). Proteins that bind and/or use NAD(H) as a co-substrate (such as, CtBP and PARPs/Sirtuins respectively) are known to induce changes in chromatin structure and transcriptional profiles. In fact, their ability to sense perturbations in NAD(H) levels has been implicated in their roles in development, stress responses, metabolic homeostasis, reproduction and aging or age-related diseases. It is also becoming increasingly clear that both the levels/activities of these proteins and the availability of NAD(H) are equally important. Here we discuss the pivotal role of NAD(H) in controlling the functions of some of these proteins, the functional interplay between them and physiological implications during calorie restriction, energy homeostasis, circadian rhythm and aging.

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Anterograde and retrograde transport of neutral sphingomyelinase-2 between the Golgi and the plasma membrane.

Publication Date: 2010 Aug 14 PMID: 20713176
Authors: Milhas, D. - Clarke, C. J. - Idkowiak-Baldys, J. - Canals, D. - Hannun, Y. A.
Journal: Biochim Biophys Acta

The activation of neutral sphingomyelinase-2 (nSMase2) and consequent ceramide production are implicated in many stress-induced signaling pathways. Trafficking of nSMase2 from the Golgi compartment to the plasma membrane (PM) in response to signaling stimuli has been described. However, the precise mechanisms of transport remain unknown. This study aimed to investigate the trafficking of nSMase2 between the Golgi and the PM. We show here that V5-nSMase2 localizes at the PM and Golgi in MCF-7 cells and confirm relocalization of nSMase2 to the PM at confluence. Although cycloheximide (CHX) treatment partially inhibited the Golgi localization of GFP-nSMase2, recovery of GFP-nSMase2 to an intracellular compartment was still observed after photobleaching. Moreover, in the presence of CHX, GFP- and V5-nSMase2 co-localized with endosomal/recycling markers. In HEK293 cells, activation of either protein kinase C-alpha or betaII, with the phorbol ester PMA led to relocalization of both wild-type and inactive nSMase2 to the pericentrion, a PKC-dependent subset of recycling endosomes. Finally, inhibition of nSMase2 endocytosis by K+depletion reduced the intracellular pool of nSMase2 and increased nSMase2 activity resulting in elevated ceramide levels. Altogether, these results suggest that nSMase2 traffics from the Golgi to the PM as a membrane protein en route to the cell surface and recycles back to the Golgi through the endosomal/recycling compartment. Moreover, the recycling of nSMase2 from the PM is important for its catalytic regulation.

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Aldosterone: A cardiovascular risk factor?

Publication Date: 2010 Aug 13 PMID: 20713154
Authors: Funder, J. W. - Reincke, M.
Journal: Biochim Biophys Acta

The hormone aldosterone has a well-recognized physiological role in epithelial fluid and electrolyte homeostasis, and more recently defined pathophysiological roles in the cardiovascular system. The term "risk factor" implies an active role in pathophysiology, with levels lower (e.g. HDL) or higher (e.g. LDL, BP) than normal contributing to an increased likelihood of morbidity and/or mortality. In this regard, primary aldosteronism represents a classic illustration of aldosterone as a cardiovascular risk factor. In this syndrome of (relatively) autonomous aldosterone secretion, the effects of elevated hormone levels are on a range of organs and tissues-the heart, blood vessels and brain, inter alia. In other cardiovascular disorders (e.g. CCF, EH) while an elevation of aldosterone levels is often regarded as a risk factor, it is more correctly a response to the severity of disease (or to treatment intervention), rather than necessarily a risk factor with a primary role in disease progression. An enduring enigma relevant to any discussion of aldosterone as a risk factor is that very high levels of aldosterone in response to chronic sodium deficiency have homeostatic (and protective of cardiovascular) functions, while the considerably lower levels commonly seen in primary aldosteronism are incontrovertibly damaging. A final section of the paper will thus propose a mechanism which might solve this enigma, based on the commonalities-and a single crucial difference-in the factors stimulating the secretion of aldosterone and endogenous ouabain from the zona glomerulosa of the adrenal gland.

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High-molecular weight hyaluronan reduced renal PKC activation in genetically diabetic mice.

Publication Date: 2010 Aug 13 PMID: 20713153
Authors: Campo, G. M. - Avenoso, A. - Micali, A. - Nastasi, G. - Squadrito, F. - Altavilla, D. - Bitto, A. - Polito, F. - Rinaldi, M. G. - Calatroni, A. - D'Ascola, A. - Campo, S.
Journal: Biochim Biophys Acta

The cluster determinant (CD44) seems to play a key role in tissues injured by diabetes type 2. CD44 stimulation activates the protein kinase C (PKC) family which in turn activates the transcriptional nuclear factor kappa B (NF-kappaB) responsible for the expression of the inflammation mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-18 (IL-18), inducible nitric oxide synthase (iNOS), and matrix metalloproteinases (MMPs). Regulation of CD44 interaction with its ligands depends greatly upon PKC. We investigated the effect of the treatment with high-molecular weight hyaluronan (HA) on diabetic nephropathy in genetically diabetic mice. BKS.Cg-m+/+Lepr(db) mice had elevated plasma insulin from 15days of age and high blood sugar levels at 4weeks. The severe nephropathy that developed was characterized by a marked increased in CD44 receptors, protein kinase C betaI, betaII, and epsilon (PKC(betaI), PKC(betaII), and PKCepsilon) mRNA expression and the related protein products in kidney tissue. High levels of mRNA and related protein levels were also detected in the damaged kidney for NF-kappaB, TNF-alpha, IL-6, IL-18, MMP-7, and iNOS. Chronic daily administration of high-molecular mass HA for 2weeks significantly reduced CD44, PKC(betaI), PKC(betaII), and PKCalpha gene expression and the related protein production in kidney tissue and TNF-alpha, IL-6, IL-18, MMP-7, and iNOS expression and levels also decreased. Histological analysis confirmed the biochemical data. However, blood parameters of diabetes were unchanged. These results suggest that the CD44 and PKC play an important role in diabetes and interaction of high-molecular weight HA with these proteins may reduce inflammation and secondary pathologies due to this disease.

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Stem cell potential in Parkinson's disease and molecular factors for the generation of dopamine neurons.

Publication Date: 2010 Aug 14 PMID: 20713152
Authors: Kim, H. J.
Journal: Biochim Biophys Acta

Parkinson's disease (PD) involves the loss of dopamine (DA) neurons, making it the most expected neurodegenerative disease to be treated by cell replacement therapy. Stem cells are a promising source for cell replacement therapy due to their ability to self-renew and their pluripotency/multipotency that allows them to generate various types of cells. However, it is challenging to derive midbrain DA neurons from stem cells. Thus, in this review, I will discuss the molecular factors that are known to play critical roles in the generation and survival of DA neurons. The developmental process of DA neurons and functions of extrinsic soluble factors and homeodomain proteins, forkhead box proteins, proneural genes, Nurr1 and genes involved in epigenetic control are discussed. In addition, different types of stem cells that have potential for use in future cell replacement therapy are reviewed.

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Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae.

Publication Date: 2010 Aug 14 PMID: 20713131
Authors: Huang, X. - Morse, L. R. - Xu, Y. - Zahradka, J. - Sychrova, H. - Stashenko, P. - Fan, F. - Battaglino, R. A.
Journal: Biochim Biophys Acta

BACKGROUND: NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes. METHODS: The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth. RESULTS: Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4M NaCl, suggesting that these residues are also essential for antiporter activity. CONCLUSIONS: Evolutionarily conserved amino acids are required for full antiporter function. GENERAL SIGNIFICANCE: Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.

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Apoptogenic factors released from mitochondria.

Publication Date: 2010 Aug 14 PMID: 20713095
Authors: Vaux, D. L.
Journal: Biochim Biophys Acta

When cells kill themselves, they usually do so by activating mechanisms that have evolved specifically for that purpose. These mechanisms, which are broadly conserved throughout the metazoa, involve two processes: activation in the cytosol of latent cysteine proteases (termed caspases), and disruption of mitochondrial functions. These processes are linked in a number of different ways. While active caspases can cleave proteins in the mitochondrial outer membrane, and cleave and thereby activate certain pro-apoptotic members of the Bcl-2 family, proteins released from the mitochondria can trigger caspase activation and antagonise IAP family proteins. This review will focus on the pro-apoptotic molecules that are released from the mitochondria of cells endeavouring to kill themselves.

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Specific inhibition of pathogenic receptor tyrosine kinase activation by its transmembrane domain.

Publication Date: 2010 Aug 14 PMID: 20713021
Authors: He, L. - Shobnam, N. - Hristova, K.
Journal: Biochim Biophys Acta

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) are believed to be important players in RTK signal transduction. However, the degree of specificity and promiscuity of RTK TM domain lateral interactions in mammalian membranes has not been assessed in detail in the literature. A technique to probe the occurrence of interactions between TM domains and their biological significance is to evaluate the propensity for formation of heterodimers of a full-length RTK and its TM domain. Here we examine if specific inhibition of two RTK pathogenic mutants, Neu/V664E and FGFR3/A391E, can be achieved by the TM domains of Neu, Neu/V664E, FGFR3 and FGFR3/A391E. We show that the TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E, while the wild-type Neu TM domain does not. In addition, Neu/V664E TM domain does not affect the phosphorylation levels of full-length FGFR3/A391E. The results suggest that TM domain peptides could be exploited in the future for the development of specific inhibitors of mutant RTKs.

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Dimerization and ligand binding affect the structure network of A(2A) adenosine receptor.

Publication Date: 2010 Aug 14 PMID: 20713020
Authors: Fanelli, F. - Felline, A.
Journal: Biochim Biophys Acta

G protein Coupled Receptors (GPCRs) are allosteric proteins whose functioning fundamentals are the communication between the two poles of the helix bundle. The representation of GPCR structures as networks of interacting amino acids can be a meaningful way to decipher the impact of ligand and of dimerization/oligomerization on the molecular communication intrinsic to the protein fold. In this study, we predicted likely homodimer architectures of the A(2A)R and investigated the effects of dimerization on the structure network and the communication paths of the monomeric form. The results of this study emphasize the roles of helix 1 in A(2A)R dimerization and of highly conserved amino acids in helices 1, 2, 6 and 7 in maintaining the structure network of the A(2A)R through a persistent hub behavior as well as in the information flow between the extracellular and intracellular poles of the helix bundle. The arginine of the conserved E/DRY motif, R3.50, is not involved in the communication paths but participates in the structure network as a stable hub, being permanently linked to E6.30 like in the inactive states of rhodopsin. A(2A)R dimerization affects the communication networks intrinsic to the receptor fold in a way dependent on the dimer architecture. Certain architectures retain the most recurrent communication paths with respect to the monomeric antagonist-bound form but enhancing path numbers and frequencies, whereas some others impair ligand-mediated communication networks. Ligand binding affects the network as well. Overall, the communication network that pertains to the functional dynamics of a GPCR is expected to be influenced by ligand functionality, oligomeric order and architecture of the supramolecular assembly.

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Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes.

Publication Date: 2010 Aug 14 PMID: 20713019
Authors: Basso, L. G. - Rodrigues, R. Z. - Naal, R. M. - Costa-Filho, A. J.
Journal: Biochim Biophys Acta

Primaquine (PQ) is a potent therapeutic agent used in the treatment of malaria and its mechanism of action still lacks a more detailed understanding at a molecular level. In this context, we used differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), and electron spin resonance (ESR) to investigate the effects of PQ on the lipid phase transition, acyl chain dynamics, and on volumetric properties of lipid model membranes. DSC thermograms revealed that PQ stabilizes the fluid phase of the lipid model membranes and interacts mainly with the lipid headgroups. This result was revealed by the great effect on the pretransition of phosphatidylcholines and the destabilization of the inverted hexagonal phase of a phosphatidylethanolamine bilayer. Spin probes located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQ is effective in changing the acyl chain ordering and dynamics of the whole chain of dimyristoylphosphatidylcholine (DMPC) phospholipid in the rippled gel phase. The combined ESR and PPC results revealed that the slight DMPC volume changes at the main phase transition induced by the presence of PQ is probably due to a less dense lipid gel phase. At physiological pH, the cationic amphiphilic PQ strongly interacts with the lipid headgroup region of the bilayers, causing considerable disorganization in the hydrophobic core. These results shed light on the molecular mechanism of primaquine-lipid interaction, which may be useful in the understanding of the complex mechanism of action and/or the adverse effects of this antimalarial drug.

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Proteomics of skeletal muscle glycolysis.

Publication Date: 2010 Aug 12 PMID: 20709194
Authors: Ohlendieck, K.
Journal: Biochim Biophys Acta

Glycolysis represents one of the best-understood and most ancient metabolic pathways. In skeletal muscle fibres, energy for contraction is supplied by adenosine triphosphate via anaerobic glycolysis, the phosphocreatine shuttle and oxidative phosphorylation. In this respect, the anaerobic glycolytic pathway supports short duration performances of contractile tissues of high intensity. The catalytic elements associated with glycolysis are altered during development, muscle differentiation, physiological adaptations and many pathological mechanisms, such as muscular dystrophy, diabetes mellitus and age-related muscle weakness. Although gel electrophoresis-based proteomics is afflicted with various biological and technical problems, it is an ideal analytical tool for studying the abundant and mostly soluble enzymes that constitute the glycolytic system. This review critically examines the proteomic findings of recent large-scale studies of glycolytic enzymes and associated components in normal, transforming and degenerating muscle tissues. In the long term, proteins belonging to the glycolytic pathway may be useful as biomarkers of muscle adaptations and pathophysiological mechanisms and can be employed to improve diagnostics and in the identification of novel therapeutic targets in neuromuscular disorders.

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Inhibitory activities of the heterotrimers formed from two alpha-type phospholipase A(2) inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation.

Publication Date: 2010 Aug 12 PMID: 20709193
Authors: Nishida, M. - Okamoto, M. - Ohno, A. - Okumura, K. - Hayashi, K. - Ikeda, K. - Inoue, S.
Journal: Biochim Biophys Acta

alpha-type phospholipase A(2) inhibitory protein (PLIalpha) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIalpha, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIalpha-LP, is homologous to GbPLIalpha, but it does not show any inhibitory activity against PLA(2)s. When a mixture of denaturant-treated monomeric forms of GbPLIalpha and EqPLIalpha-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIalpha and EqPLIalpha-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIalpha and the recombinant chimeric EqPLIalpha-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLIalpha, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIalpha-LP and the recombinant chimeric GbPLIalpha, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLIalpha-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIalpha were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIalpha with a single amino acid substitution, GbPLIalpha(K28E).

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Gene regulation by SMAR1: Role in cellular homeostasis and cancer.

Publication Date: 2010 Aug 13 PMID: 20709157
Authors: Malonia, S. K. - Sinha, S. - Lakshminarasimhan, P. - Singh, K. - Jalota-Badhwar, A. - Rampalli, S. - Kaul-Ghanekar, R. - Chattopadhyay, S.
Journal: Biochim Biophys Acta

Changes in the composition of nuclear matrix associated proteins contribute to alterations in nuclear structure, one of the major phenotypes of malignant cancer cells. The malignancy-induced changes in this structure lead to alterations in chromatin folding, the fidelity of genome replication and gene expression programs. The nuclear matrix forms a scaffold upon which the chromatin is organized into periodic loop domains called matrix attachment regions (MAR) by binding to various MAR binding proteins (MARBPs). Aberrant expression of MARBPs modulates the chromatin organization and disrupt transcriptional network that leads to oncogenesis. Dysregulation of nuclear matrix associated MARBPs has been reported in different types of cancers. Some of these proteins have tumor specific expression and are therefore considered as promising diagnostic or prognostic markers in few cancers. SMAR1 (scaffold/matrix attachment region binding protein 1), is one such nuclear matrix associated protein whose expression is drastically reduced in higher grades of breast cancer. SMAR1 gene is located on human chromosome 16q24.3 locus, the loss of heterozygosity (LOH) of which has been reported in several types of cancers. This review elaborates on the multiple roles of nuclear matrix associated protein SMAR1 in regulating various cellular target genes involved in cell growth, apoptosis and tumorigenesis.

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The simulation of interquinone charge transfer in a bacterial photoreaction center highlights the central role of a hydrogen-bonded non-heme iron complex.

Publication Date: 2010 Aug 13 PMID: 20709018
Authors: Burggraf, F. - Koslowski, T.
Journal: Biochim Biophys Acta

We consider electron transfer between the quinones Q(A) and Q(B), one of the final steps in the photoinduced charge separation in the photoreaction center of Rhodobacter sphaeroides. The system is described by a model with atomic resolution using classical force fields and a carefully parameterized tight-binding Hamiltonian. The rates estimated for direct interquinone charge transfer hopping involving a non-heme complex bridging the quinones and superexchange based on the geometry of the photochemically inactive dark state are orders of magnitude smaller than those obtained experimentally. Only if the iron complex is attached to both quinones via hydrogen bonds - as characteristic of the charge transfer active light state - the computed rate for superexchange involving the histidine ligands of the complex will become comparable to the experimental value of k(CT)=10(5) s(-1).

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The Aurora-A/TPX2 complex: A novel oncogenic holoenzyme?

Publication Date: 2010 Aug 12 PMID: 20708655
Authors: Asteriti, I. A. - Rensen, W. M. - Lindon, C. - Lavia, P. - Guarguaglini, G.
Journal: Biochim Biophys Acta

The Aurora-A kinase regulates cell division by phosphorylating multiple downstream targets in the mitotic apparatus. Aurora-A is frequently overexpressed in tumor cells and it is therefore regarded as a novel candidate target in anti-cancer therapy. Its actual contribution to cell transformation, however, is not entirely clarified; furthermore, its transforming ability has been found to vary broadly depending on the systems and experimental conditions in which it was assayed. This variability suggests that Aurora-A overexpression requires the concomitant deregulation of partner factor(s) to fully elicit its oncogenic potential. Molecular and structural studies indicate that the full activation and correct mitotic localisation of Aurora-A require its interaction with the spindle regulator TPX2. In this review we propose a brief reappraisal of Aurora-A intrinsic oncogenic features. We then present literature screening data indicating that TPX2 is also overexpressed in many tumor types, and, furthermore, that Aurora-A and TPX2 are frequently co-overexpressed. We therefore propose that the association of Aurora-A and TPX2 gives rise to a novel functional unit with oncogenic properties. We also suggest that some of the roles that are conventionally attributed to Aurora-A in cell transformation and tumorigenesis could in fact be a consequence of the oncogenic activation of this unit.

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Validating cancer drug targets through chemical genetics.

Publication Date: 2010 Aug 12 PMID: 20708654
Authors: Burkard, M. E. - Jallepalli, P. V.
Journal: Biochim Biophys Acta

Targeted therapies for cancer promise to revolutionize treatment by specifically inactivating pathways needed for the growth of tumor cells. The most prominent example of such therapy is imatinib (Gleevec), which targets the Bcr-Abl kinase and provides an effective low-toxicity treatment for chronic myelogenous leukemia. This success has spawned myriad efforts to develop similarly targeted drugs for other cancers. Unfortunately, the high expectations of these efforts have not yet been realized, likely due to the genetic diversity among and within tumors, as well as the complex and largely unpredictable interactions of drug-like compounds with innumerable targets that affect cellular and organismal metabolism. While improvements in sequencing technologies are beginning to address the first problem, solving the second problem requires methods for linking specific features of the cancer genome to their optimally targeted therapies. One approach, referred to as chemical genetics, accomplishes this by genetic control of chemical susceptibility. Chemical genetics is a crucial tool for the rational development of cancer drugs.

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Insulin stimulation of PKCdelta triggers its rapid degradation via the ubiquitin-proteasome pathway.

Publication Date: 2010 Aug 12 PMID: 20708645
Authors: Brand, C. - Horovitz-Fried, M. - Inbar, A. - Tamar-Brutman-Barazani - Brodie, C. - Sampson, S. R.
Journal: Biochim Biophys Acta

Insulin rapidly upregulates protein levels of PKCdelta in classical insulin target tissues skeletal muscle and liver. Insulin induces both a rapid increase in de novo synthesis of PKCdelta protein. In this study we examined the possibility that insulin may also inhibit degradation of PKCdelta. Experiments were performed on L6 skeletal muscle myoblasts or myotubes in culture. Phorbol ester (PMA)- and insulin-induced degradation of PKCdelta were abrogated by proteasome inhibition. Both PMA and insulin induced ubiquitination of PKCdelta, but not of that PKCalpha or PKCepsilon and increased proteasome activity within 5min. We examined the role of tyrosine phosphorylation of PKCdelta in targeting PKCdelta for degradation by the ubiquitin-proteasome pathway. Transfection of cells with PKCdeltaY(311)F, which is not phosphorylated, resulted in abolition of insulin-induced ubiquitination of PKCdelta and increase in proteasome activity. We conclude that insulin induces degradation of PKCdelta via the ubiquitin-proteasome system, and that this effect requires phosphorylation on specific tyrosine residues for targeting PKCdelta for degradation by the ubiquitin-proteasome pathway. These studies provide additional evidence for unique effects of insulin on regulation of PKCdelta protein levels.

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The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells.

Publication Date: 2010 Aug 12 PMID: 20708644
Authors: Sanidas, I. - Kotoula, V. - Ritou, E. - Daans, J. - Lenz, C. - Mairhofer, M. - Daniilidou, M. - Kolbus, A. - Kruft, V. - Ponsaerts, P. - Nikolakaki, E.
Journal: Biochim Biophys Acta

SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.

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Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.

Publication Date: 2010 Aug 12 PMID: 20708643
Authors: Bradamante, S. - Villa, A. - Versari, S. - Barenghi, L. - Orlandi, I. - Vai, M.
Journal: Biochim Biophys Acta

A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells ( approximately 40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).

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Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-Differential sensitivity to calcium and phosphorylation events.

Publication Date: 2010 Aug 11 PMID: 20708598
Authors: Carrichon, L. - Picciocchi, A. - Debeurme, F. - Defendi, F. - Beaumel, S. - Jesaitis, A. J. - Dagher, M. C. - Stasia, M. J.
Journal: Biochim Biophys Acta

NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b(558), the redox core, is assembled with cytosolic p47(phox), p67(phox), p40(phox), and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O(2). Moreover, replacement of this loop by the Nox4-D-loop (D-loop(Nox4)-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca(2+) influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b(558) was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loop(Nox4)-Nox2 mutant may come from a change of responsiveness to intracellular Ca(2+) level and to phosphorylation events during oxidase activation. Finally the D-loop(Nox4)-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.

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ORF8a of SARS-CoV forms an ion channel: Experiments and molecular dynamics simulations.

Publication Date: 2010 Aug 12 PMID: 20708597
Authors: Chen, C. C. - Kruger, J. - Sramala, I. - Hsu, H. J. - Henklein, P. - Chen, Y. M. - Fischer, W. B.
Journal: Biochim Biophys Acta

ORF8a protein is 39 residues long and contains a single transmembrane domain. The protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers that form cation-selective ion channels with a main conductance level of 8.9+/-0.8pS at elevated temperature (38.5 degrees C). Computational modeling studies including multi nanosecond molecular dynamics simulations in a hydrated POPC lipid bilayer are done with a 22 amino acid transmembrane helix to predict a putative homooligomeric helical bundle model. A structural model of a pentameric bundle is proposed with cysteines, serines and threonines facing the pore.

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Mesenchymal stem cells stimulate angiogenesis in a murine xenograft model of A549 human adenocarcinoma through an LPA1 receptor-dependent mechanism.

Publication Date: 2010 Aug 11 PMID: 20708100
Authors: Jeon, E. S. - Lee, I. H. - Heo, S. C. - Shin, S. H. - Choi, Y. J. - Park, J. H. - Park, D. Y. - Kim, J. H.
Journal: Biochim Biophys Acta

Carcinoma-associated fibroblasts play a key role in tumorigenesis and metastasis by providing a tumor-supportive microenvironment. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to carcinoma-associated fibroblasts expressing alpha-smooth muscle actin, vascular endothelial growth factor, and stromal cell-derived factor-1. A549 conditioned medium-induced differentiation of hASCs to carcinoma-associated fibroblasts was completely abrogated by treatment of hASCs with Ki16425, a lysophosphatidic acid receptor antagonist, or knockdown of lysophosphatidic acid receptor 1 (LPA(1)) expression in hASCs with small interfering RNA or lentiviral short hairpin RNA. Using a murine xenograft transplantation model of A549 cells, we showed that co-transplantation of hASCs with A549 cells stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts in vivo. Knockdown of LPA(1) expression in hASCs abrogated hASCs-stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts. Moreover, A549 conditioned medium-treated hASCs stimulated tube formation of human umbilical vein endothelial cells by LPA(1)-dependent secretion of vascular endothelial growth factor. These results suggest that A549 cells induce in vivo differentiation of hASCs to carcinoma-associated fibroblasts, which play a key role in tumor angiogenesis within tumor microenvironment, through an LPA-LPA(1)-mediated paracrine mechanism.

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Specialized pro-resolving lipid mediators in the inflammatory response: An update.

Publication Date: 2010 Aug 10 PMID: 20708099
Authors: Bannenberg, G. - Serhan, C. N.
Journal: Biochim Biophys Acta

A new genus of specialized pro-resolving mediators (SPM) which include several families of distinct local mediators (lipoxins, resolvins, protectins, and maresins) are actively involved in the clearance and regulation of inflammatory exudates to permit restoration of tissue homeostasis. Classic lipid mediators that are temporally regulated are formed from arachidonic acid, and novel local mediators were uncovered that are biosynthesized from omega-3 poly-unsaturated fatty acids, such as eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid. The biosynthetic pathways for resolvins are constituted by fatty acid lipoxygenases and cyclooxygenase-2 via transcellular interactions established by innate immune effector cells which migrate from the vasculature to inflamed tissue sites. SPM provide local control over the execution of an inflammatory response towards resolution, and include recently recognized actions of SPM such as tissue protection and host defense. The structural families of the SPM do not resemble classic eicosanoids (PG or LT) and are novel structures that function uniquely via pro-resolving cellular and molecular targets. The extravasation of inflammatory cells expressing SPM biosynthetic routes are matched by the temporal provision of essential fatty acids from circulation needed as substrate for the formation of SPM. The present review provides an update and overview of the biosynthetic pathways and actions of SPM, and examines resolution as an integrated component of the inflammatory response and its return to homeostasis via biochemically active resolution mechanisms.

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gamma2-Adaptin is functioning in the late endosomal sorting pathway and interacts with ESCRT-I and -III subunits.

Publication Date: 2010 Aug 10 PMID: 20708039
Authors: Doring, T. - Gotthardt, K. - Stieler, J. - Prange, R.
Journal: Biochim Biophys Acta

gamma2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that gamma2-adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit gamma2-adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed gamma2-adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess gamma2-adaptin acts by accelerating lysosomal Gag destruction. Consistently, depletion of gamma2-adaptin avoids Gag routing to the lysosome and increases viral production. Functional studies with natural MVB cargoes support a role of gamma2-adaptin in MVB-to-lysosome transition. Furthermore, we show that different members of the endosomal sorting complex required for transport (ESCRT) that drive sorting from endosomes to lysosomes are sequestered upon gamma2-adaptin overexpression. If sequestered irreversibly, they are targeted to enhanced lysosomal degradation. The participation of gamma2-adaptin in MVB sorting is further suggested by our finding that it specifically interacts with the ESCRT subunits Vps28 and CHMP2A. These observations identify gamma2-adaptin as a critical factor in MVB trafficking, which likely is involved in endosome-to-lysosome maturation.

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Expression and regulation of a novel identified TNFAIP8 family is associated with diabetic nephropathy.

Publication Date: 2010 Aug 8 PMID: 20699119
Authors: Zhang, S. - Zhang, Y. - Wei, X. - Zhen, J. - Wang, Z. - Li, M. - Miao, W. - Ding, H. - Du, P. - Zhang, W. - He, M. - Yi, F.
Journal: Biochim Biophys Acta

Tumor necrosis factor-alpha-inducible protein 8 (TNFAIP8) family are very recently identified proteins which share considerable sequence homology to regulate cellular and immune homeostasis. However, it is unknown whether TNFAIP8 family is expressed in the kidney and contributes to the regulation of renal functions. Therefore, the present study was designed to characterize the members of TNFAIP8 family in the kidney and to explore their possible roles in the development and progression of diabetic nephropathy. By RT-PCR and Western blot analyses, we found that all members of TNFAIP8 family were detected in the kidney. TNFAIP8 and TIPE2 expression was significantly increased in glomeruli from streptozotocin (STZ)-induced diabetic rats, and this upregulation was further confirmed in renal biopsies of diabetic patients. In in vitro study, TNFAIP8 was upregulated in response to high glucose in mesangial cells rather than podocytes. Moreover, a direct correlation was observed between expression of TNFAIP8 and mesangial cell proliferation and this regulation was associated with NADPH oxidase-mediated signaling pathway. However, we failed to observe the upregulation of TIPE2 in both mesangial cells and podocytes in response to high glucose. In conclusion, the present study addressed the role of TNFAIP8 family in diabetic nephropathy. These findings for the first time demonstrate that TNFAIP8 is one of critical components of a signal transduction pathway that links mesangial cell proliferation to diabetic renal injury.

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Spectroscopic studies of molecular organization of antibiotic amphotericin B in monolayers and dipalmitoylphosphatidylcholine lipid multibilayers.

Publication Date: 2010 Aug 10 PMID: 20699086
Authors: Gagos, M. - Arczewska, M.
Journal: Biochim Biophys Acta

Amphotericin B (AmB) is considered the gold-standard in the treatment of serious systemic mycoses despite its numerous adverse effects. Both the mechanism of antifungal action and the toxicity of this drug are dependent on its molecular organization. The effect of AmB on the organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) was studied with application of the Langmuir-Blodgett technique and ATR-FTIR spectroscopy. The aim of this research was to analyze the physical interactions leading to the formation of aggregated forms of AmB molecules in one-component monolayers and lipid multibilayers. Analysis of FTIR spectra of two-component multibilayers suggests the possibility the mutual reorientation of the amino-sugar moiety (mycosamine) and macrolide ring. This effect may be significant in the explanation of the aggregation processes of AmB in biological systems.

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Different altered pattern expression of genes related to apoptosis in isolated methylmalonic aciduria cblB type and combined with homocystinuria cblC type.

Publication Date: 2010 Aug 6 PMID: 20696242
Authors: Jorge-Finnigan, A. - Gamez, A. - Perez, B. - Ugarte, M. - Richard, E.
Journal: Biochim Biophys Acta

An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders involved in cobalamin metabolism, including isolated methylmalonic aciduria (MMA) cblB type and MMA combined with homocystinuria (MMAHC) cblC type. Given the relevance of p38 and JNK kinases in stress-response, their activation in fibroblasts from a spectrum of patients (mut, cblA, cblB, cblC and cblE) was analyzed revealing an increased expression of the phosphorylated-forms, specially in cblB and cblC cell lines that presented the highest ROS and apoptosis levels. To gain further insight into the molecular mechanisms responsible for the enhanced apoptotic process observed in cblB and cblC fibroblasts, we evaluated the expression pattern of 84 apoptosis-related genes by quantitative real-time PCR. An elevated number of pro-apoptotic genes were overexpressed in cblC cells showing a higher rate of apoptosis compared to cblB and control samples. Additionally, apoptosis appears to be mainly triggered through the extrinsic pathway in cblC, while the intrinsic pathway was primarily activated in cblB cells. The differences observed regarding the apoptosis rate and preferred pathway between cblB and cblC patients, who both built up methylmalonic acid, might be explained by the accumulated homocysteine in the cblC group. The loss of MMACHC function in cblC patients might be partially responsible for the oxidative stress and apoptosis processes observed in these cell lines. Our results suggest that ROS production may represent a genetic modifier of the phenotype and support the potential of using antioxidants as a novel therapeutic strategy to improve the severe neurological outcome of these rare diseases.

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Functional analysis of F508del CFTR in native human colon.

Publication Date: 2010 Aug 7 PMID: 20696241
Authors: van Barneveld, A. - Stanke, F. - Tamm, S. - Siebert, B. - Brandes, G. - Derichs, N. - Ballmann, M. - Junge, S. - Tummler, B.
Journal: Biochim Biophys Acta

The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.

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Human endogenous retroviruses and multiple sclerosis: Innocent bystanders or disease determinants?

Publication Date: 2010 Aug 6 PMID: 20696240
Authors: Antony, J. M. - Deslauriers, A. M. - Bhat, R. K. - Ellestad, K. K. - Power, C.
Journal: Biochim Biophys Acta

Human endogenous retroviruses (HERVs) constitute 5-8% of human genomic DNA and are replication incompetent despite expression of individual HERV genes from different chromosomal loci depending on the specific tissue. Several HERV genes have been detected as transcripts and proteins in the central nervous system, frequently in the context of neuroinflammation. The HERV-W family has received substantial attention in large part because of associations with diverse syndromes including multiple sclerosis (MS) and several psychiatric disorders. A HERV-W-related retroelement, multiple sclerosis retrovirus (MSRV), has been reported in MS patients to be both a biomarker as well as an effector of aberrant immune responses. HERV-H and HERV-K have also been implicated in MS and other neurological diseases but await delineation of their contributions to disease. The HERV-W envelope-encoded glycosylated protein, syncytin-1, is encoded by chromosome 7q21 locus and exhibits increased glial expression within MS lesions. Overexpression of syncytin-1 in glia induces endoplasmic reticulum stress leading to neuroinflammation and the induction of free radicals, which damage proximate cells. Syncytin-1's receptor, ASCT1 is a neutral amino acid transporter expressed on glia and is suppressed in white matter of MS patients. Of interest, antioxidants ameliorate syncytin-1's neuropathogenic effects raising the possibility of using these agents as therapeutics for neuroinflammatory diseases. Given the multiple insertion sites of HERV genes as complete and incomplete open reading frames, together with their differing capacity to be expressed and the complexities of individual HERVs as both disease markers and bioactive effectors, HERV biology is a compelling area for understanding neuropathogenic mechanisms and developing new therapeutic strategies.

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Mitochondrial protein import machineries and lipids: A functional connection.

Publication Date: 2010 Aug 7 PMID: 20696129
Authors: Gebert, N. - Ryan, M. T. - Pfanner, N. - Wiedemann, N. - Stojanovski, D.
Journal: Biochim Biophys Acta

Protein trafficking and translocation are essential processes in even the simplest living cells. The compartmentalisation within eukaryotic cells places a very high demand on the fidelity of protein trafficking and translocation, since a large percentage of the cell's protein complement is inserted into, or translocated across membranes. Indeed, most mitochondrial proteins are imported from the cytosol into the organelle and reach their final destination with the assistance of versatile translocation machineries. The first components involved in mitochondrial protein import were identified about 20years ago and over the last two decades many new factors and machineries have been brought to light. However, in spite of these discoveries we still have much to explore regarding the molecular mechanisms that distinguish the different mitochondrial import pathways. In particular, an open question that requires deeper exploration is the role of lipids and lipid modifying enzymes in this process. Mitochondrial biogenesis requires the coordinated synthesis and import of both proteins and phospholipids, however, these have typically been considered as distinct research fields. Recent findings have placed phospholipids at the forefront of research dealing with mitochondrial biogenesis, in particular their role in the regulation of mitochondrial transport machineries.

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Simulations of calcium channel block by trivalent cations: Gd(3+) competes with permeant ions for the selectivity filter.

Publication Date: 2010 Aug 7 PMID: 20696128
Authors: Malasics, A. - Boda, D. - Valisko, M. - Henderson, D. - Gillespie, D.
Journal: Biochim Biophys Acta

Current through L-type calcium channels (Ca(V)1.2 or dihydropyridine receptor) can be blocked by micromolar concentrations of trivalent cations like the lanthanide gadolinium (Gd(3+)). It has been proposed that trivalent block is due to ions competing for a binding site in both the open and closed configuration, but possibly with different trivalent affinities. Here, we corroborate this general view of trivalent block by computing conductance of a model L-type calcium channel. The model qualitatively reproduces the Gd(3+) concentration dependence and the effect that substantially more Gd(3+) is required to produce similar block in the presence of Sr(2+) (compared to Ba(2+)) and even more in the presence of Ca(2+). Trivalent block is explained in this model by cations binding in the selectivity filter with the charge/space competition mechanism. This is the same mechanism that in the model channel governs other selectivity properties. Specifically, selectivity is determined by the combination of ions that most effectively screen the negative glutamates of the protein while finding space in the midst of the closely packed carboxylate groups of the glutamate residues.

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Conformational and biochemical characterization of a rat epididymis-specific lipocalin 12 expressed in Escherichia coli.

Publication Date: 2010 Aug 6 PMID: 20692383
Authors: Peng, Y. - Liu, J. - Liu, Q. - Yao, Y. - Guo, C. - Zhang, Y. - Lin, D.
Journal: Biochim Biophys Acta

Lipocalin 12 (Lcn12) is a recently identified epididymis-specific protein that might play a significant physiological role in male reproduction. However, the detailed structure and function of Lcn12 remain to be determined. In the present work, we cloned, expressed, and purified the rat Lcn12 (rLcn12) protein in Escherichia coli, introduced the Cys176Ala substitution to eliminate the aggregation problem associated with the wild-type protein. Homology modeling results demonstrated that rLcn12 adopted an eight-stranded, antiparallel beta-barrel conformation containing a conserved disulfide bond between Cys98 and Cys203, which was in accordance with the physicochemical properties elucidated by a combination of mass, circular dichroism, and nuclear magnetic resonance spectrometry. The purified rLcn12 protein exhibited a high binding affinity for all-trans retinoic acid in fluorescence titration experiments, implying that rLcn12 could be involved in retinoic acid transport in the epididymis.

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Lipin - The bridge between hepatic glycerolipid biosynthesis and lipoprotein metabolism.

Publication Date: 2010 Aug 6 PMID: 20692363
Authors: Khalil, M. B. - Blais, A. - Figeys, D. - Yao, Z.
Journal: Biochim Biophys Acta

Growing evidence links the three mammalian lipin proteins, i.e., lipin-1, lipin-2 and lipin-3, to metabolic and cardiovascular diseases such as noninsulin-dependent diabetes mellitus and atherosclerosis. Lipin proteins play a dual function in lipid metabolism by acting as phosphatidate phosphatase (PAP) enzymes and as transcriptional regulators. Genetic variants within the human LPIN1 and LPIN2 genes are associated with metabolic syndromes. The fatty liver dystrophy (fld) mice carrying mutations within the Lpin1 gene display life-long deficiency in adipogenesis, insulin resistance, neonatal hepatosteatosis and hypertriglyceridemia, as well as increased atherosclerosis susceptibility. Cell culture studies show that hepatic lipin-1 expression is selectively stimulated by glucocorticoids and repressed by insulin, and its subcellular localization governs the assembly and secretion of very low density lipoproteins (VLDL). In noninsulin-dependent diabetes, glucocorticoid signals lead to dyslipidemia characterized by overproduction of VLDL and atherogenic remnants. This puts lipin-1 as a key integrator of hormonal signals to the liver in diabetic dyslipidemia. This review summarizes the current understanding of the role that hepatic lipin-1 plays in the synthesis, storage and compartmentalization of glycerolipids, and highlights the lipid metabolic consequences associated with dysregulated lipin expression.

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The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-alpha production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice.

Publication Date: 2010 Aug 6 PMID: 20692339
Authors: Shibata, T. - Nagata, K. - Kobayashi, Y.
Journal: Biochim Biophys Acta

During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-alpha production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24h. There were increases in the numbers of neutrophils and late apoptotic ones at 24h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-alpha production at 36h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12h decreased the numbers of neutrophils and late apoptotic ones at 24h, and thereafter TNF-alpha production at 36h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-alpha production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses.

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The blood-brain barrier, chemokines and multiple sclerosis.

Publication Date: 2010 Aug 6 PMID: 20692338
Authors: Holman, D. W. - Klein, R. S. - Ransohoff, R. M.
Journal: Biochim Biophys Acta

The infiltration of leukocytes into the central nervous system (CNS) is an essential step in the neuropathogenesis of multiple sclerosis (MS). Leukocyte extravasation from the bloodstream is a multistep process that depends on several factors including fluid dynamics within the vasculature and molecular interactions between circulating leukocytes and the vascular endothelium. An important step in this cascade is the presence of chemokines on the vascular endothelial cell surface. Chemokines displayed along the endothelial lumen bind chemokine receptors on circulating leukocytes, initiating intracellular signaling that culminates in integrin activation, leukocyte arrest, and extravasation. The presence of chemokines at the endothelial lumen can help guide the movement of leukocytes through peripheral tissues during normal immune surveillance, host defense or inflammation. The expression and display of homeostatic or inflammatory chemokines therefore critically determine which leukocyte subsets extravasate and enter the peripheral tissues. Within the CNS, however, infiltrating leukocytes that cross the endothelium face additional boundaries to parenchymal entry, including the abluminal presence of localizing cues that prevent egress from perivascular spaces. This review focuses on the differential display of chemokines along endothelial surfaces and how they impact leukocyte extravasation into parenchymal tissues, especially within the CNS. In particular, the display of chemokines by endothelial cells of the blood brain barrier may be altered during CNS autoimmune disease, promoting leukocyte entry into this immunologically distinct site. Recent advances in microscopic techniques, including two-photon and intravital imaging have provided new insights into the mechanisms of chemokine-mediated capture of leukocytes within the CNS.

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Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains.

Publication Date: 2010 Aug 6 PMID: 20692337
Authors: Davern, S. - Murphy, C. L. - O'Neill, H. - Wall, J. S. - Weiss, D. T. - Solomon, A.
Journal: Biochim Biophys Acta

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

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Lipid chain branching at the iso- and anteiso-positions in complex chlamydia membranes: A molecular dynamics study.

Publication Date: 2010 Aug 6 PMID: 20692231
Authors: Lim, J. B. - Klauda, J. B.
Journal: Biochim Biophys Acta

Membranes in the intracellular eubacterial parasite Chlamydia trachomatis consist of the elementary body (EB) and reticular body (RB), and contain methyl branches at the iso- and anteiso-positions for some phospholipid chains. Acyl chain branching is the focus of this study. Molecular dynamics simulations were used to study bilayers of 1-13-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (13-MpPPC), 1-14-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (14-MpPPC), and diphytanoylphosphatidylcholine (DPhPC). These three membranes were simulated at 323K and simulations of DPhPC at 298K were also performed for better comparison to existing experimental data. Two simulations of representative EB and RB membranes of C. trachomatis composed of nine different lipid components were performed at 310.15K, to accurately reflect compositions determined by experiment and physiological conditions. Based on nearly 0.5mus of simulation data, we report that branching increases average lipid surface area, lateral elastic moduli, and lipid axial relaxation times, while decreasing lipid chain order. Branching also has a distinct effect on electron density profiles. Due to their high cholesterol concentrations, the EB and RB membranes were found to have relatively high lateral elastic moduli, which may have important biological implications.

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A biophysical glance at the outer surface of the membrane transporter SGLT1.

Publication Date: 2010 Aug 6 PMID: 20692230
Authors: Tyagi, N. K. - Puntheeranurak, T. - Raja, M. - Kumar, A. - Wimmer, B. - Neundlinger, I. - Gruber, H. - Hinterdorfer, P. - Kinne, R. K.
Journal: Biochim Biophys Acta

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure-function relation. Crystallographic studies, if available, define the transmembrane segments in a "frozen" state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein. The current review presents biophysical methods that can shed light on the dynamics of transporters in the membrane. The techniques that are presented in some detail are single-molecule recognition atomic force microscopy and tryptophan scanning, which can report on the positioning of the loops and on conformational changes at the outer surface. Studies on a variety of symporters are discussed, which use gradients of sodium or protons as energy source to translocate (mainly organic) solutes against their concentration gradients into or out of the cells. Primarily, investigations of the sodium-glucose cotransporter SGLT1 are used as examples for this biophysical approach to understand transporter function.

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alpha-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression.

Publication Date: 2010 Aug 6 PMID: 20692229
Authors: Chalmeau, J. - Monina, N. - Shin, J. - Vieu, C. - Noireaux, V.
Journal: Biochim Biophys Acta

Cell-free protein synthesis is becoming a serious alternative to cell-based protein expression. Cell-free systems can deliver large amounts of cytoplasmic recombinant proteins after a few hours of incubation. Recent studies have shown that membrane proteins can be also expressed in cell-free reactions and directly inserted into phospholipid membranes. In this work, we present a quantitative method to study in real time the concurrent cell-free expression and insertion of membrane proteins into phospholipid bilayers. The pore-forming protein alpha-hemolysin, fused to the reporter protein eGFP, was used as a model of membrane protein. Cell-free expression of the toxin in solution and inside large synthetic phospholipid vesicles was measured by fluorometry and fluorescence microscopy respectively. A quartz crystal microbalance with dissipation was used to characterize the interaction of the protein with a supported phospholipid bilayer. The cell-free reaction was directly incubated onto the bilayer inside the microbalance chamber while the frequency and the dissipation signals were monitored. The presence of pores in the phospholipid bilayer was confirmed by atomic force microscopy. A model is presented which describes the kinetics of adsorption of the expressed protein on the phospholipid bilayer. The combination of cell-free expression, fluorescence microscopy and quartz crystal microbalance-dissipation is a new quantitative approach to study the interaction of membrane proteins with phospholipid bilayers.

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How different oxidation states of crystalline myoglobin are influenced by X-rays.

Publication Date: 2010 Aug 5 PMID: 20691815
Authors: Hersleth, H. P. - Andersson, K. K.
Journal: Biochim Biophys Acta

X-ray induced radiation damage of protein crystals is well known to occur even at cryogenic temperatures. Redox active sites like metal sites seem especially vulnerable for these radiation-induced reductions. It is essential to know correctly the oxidation state of metal sites in protein crystal structures to be able to interpret the structure-function relation. Through previous structural studies, we have tried to characterise and understand the reactions between myoglobin and peroxides. These reaction intermediates are relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress, and because these intermediates are similar among the haem peroxidases and oxygenases. We have in our previous studies shown that these different myoglobin states are influenced by the X-rays used. In this study, we have in detail investigated the impact that X-rays have on these different oxidation states of myoglobin. An underlying goal has been to find a way to be able to determine mostly unreduced states. We have by using single-crystal light absorption spectroscopy found that the different oxidation states of myoglobin are to a different extent influenced by the X-rays (e.g. ferric Fe(III) myoglobin is faster reduced than ferryl Fe(IV) horizontal lineO myoglobin). We observe that the higher oxidation states are not reduced to normal ferrous Fe(II) or ferric Fe(III) states, but end up in some intermediate and possibly artificial states. For ferric myoglobin, it seems that annealing of the radiation-induced/reduced state can reversibly more or less give the starting point (ferric myoglobin). Both scavengers and different dose-rates might influence to which extent the different states are affected by the X-rays. Our study shows that it is essential to do a time/dose monitoring of the influence X-rays have on each specific redox-state with spectroscopic techniques like single-crystal light absorption spectroscopy. This will determine to which extent you can collect X-ray diffraction data on your crystal before it becomes too heavily influenced/reduced by X-rays.

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Raman-assisted crystallography of biomolecules at the synchrotron: Instrumentation, methods and applications.

Publication Date: 2010 Aug 5 PMID: 20691814
Authors: McGeehan, J. E. - Bourgeois, D. - Royant, A. - Carpentier, P.
Journal: Biochim Biophys Acta

Raman spectroscopy is a powerful technique that, in recent years, has been successfully coupled to X-ray crystallography for the analysis of biological macromolecular systems. The complementarity between both techniques is illustrated at multiple stages, including sample preparation, data collection and structural interpretation with a mechanistic perspective. The current state of instrumentation is described, focusing on synchrotron based setups. Present and future applications of Raman microspectrophotometry are reviewed with reference to recent examples dealing with metallo-, photosensitive-, and redox-proteins. The added value of Raman microspectrophotometry to assess X-radiation damage is discussed, and its applicability to investigate crystalline DNA molecules is also emphasized.

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Biological effects of propionic acid in humans; metabolism, potential applications and underlying mechanisms.

Publication Date: 2010 Aug 4 PMID: 20691280
Authors: Al-Lahham, S. H. - Peppelenbosch, M. P. - Roelofsen, H. - Vonk, R. J. - Venema, K.
Journal: Biochim Biophys Acta

Undigested food is fermented in the colon by the microbiota and gives rise to various microbial metabolites. Short-chain fatty acids (SCFA), including acetic, propionic and butyric acid, are the principal metabolites produced. However, most of the literature focuses on butyrate and to a lesser extent on acetate; consequently, potential effects of propionic acid (PA) on physiology and pathology have long been underestimated. It has been demonstrated that PA lowers fatty acids content in liver and plasma, reduces food intake, exerts immunosuppressive actions and probably improves tissue insulin sensitivity. Thus increased production of PA by the microbiota might be considered beneficial in the context of prevention of obesity and diabetes type 2. The molecular mechanisms by which PA may exert this plethora of physiological effects are slowly being elucidated and include intestinal cyclooxygenase enzyme, the G-protein coupled receptors 41 and 43 and activation of the peroxisome proliferator-activated receptor gamma, in turn inhibiting the sentinel transcription factor NF-kappaB and thus increasing the threshold for inflammatory responses in general. Taken together, PA emerges as a major mediator in the link between nutrition, gut microbiota and physiology.

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Role of IL-6 trans-signaling in CCl(4) induced liver damage.

Publication Date: 2010 Aug 3 PMID: 20691261
Authors: Gewiese-Rabsch, J. - Drucker, C. - Malchow, S. - Scheller, J. - Rose-John, S.
Journal: Biochim Biophys Acta

Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. IL-6 trans-signaling has been demonstrated to operate during liver regeneration. We have developed methods to specifically block or mimic IL-6 trans-signaling. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. Using these tools we investigate the role of IL-6 trans-signaling in CCl(4) induced liver damage. Blockade of IL-6 trans-signaling during CCl(4) induced liver damage led to higher liver damage, although induction of Cyp4502E1 and thus bioactivation of CCl(4) was unchanged. Depletion of neutrophils resulted in reduced liver transaminase levels irrespective of IL-6 trans-signaling blockade. Furthermore, IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24h after CCl(4) treatment. We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl(4) induced chemical damage.

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Untranslated regions of thyroid hormone receptor beta 1 mRNA are impaired in human clear cell renal cell carcinoma.

Publication Date: 2010 Aug 3 PMID: 20691260
Authors: Master, A. - Wojcicka, A. - Piekielko-Witkowska, A. - Boguslawska, J. - Poplawski, P. - Tanski, Z. - Darras, V. M. - Williams, G. R. - Nauman, A.
Journal: Biochim Biophys Acta

Thyroid hormone receptor beta1 (TRbeta1) is a hormone-dependent transcription factor activated by 3,5,3'-l-triiodothyronine (T3). TRbeta1 functions as a tumor suppressor and disturbances of the THRB gene are frequent findings in cancer. Translational control mediated by untranslated regions (UTRs) regulates cell proliferation, metabolism and responses to cellular stress, processes that are involved in carcinogenesis. We hypothesized that reduced TRbeta1 expression in clear cell renal cell cancer (ccRCC) results from regulatory effects of TRbeta1 5' and 3'UTRs on protein translation. We determined TRbeta1 expression and alternative splicing of TRbeta1 5' and 3'UTRs in ccRCC and control tissue together with expression of the type 1 deiodinase enzyme (coded by DIO1, a TRbeta1 target gene). Tissue concentrations of T3 (which are generated in part by D1) and expression of miRNA-204 (an mRNA inhibitor for which a putative interaction site was identified in the TRbeta1 3'UTR) were also determined. TRbeta1 mRNA and protein levels were reduced by 70% and 91% in ccRCC and accompanied by absent D1 protein, a 58% reduction in tissue T3 concentration and 2-fold increase in miRNA-204. Structural analysis of TRbeta1 UTR variants indicated that reduced TRbeta1 expression may be maintained in ccRCC by posttranscriptional mechanisms involving 5'UTRs and miRNA-204. The tumor suppressor activity of TRbeta1 indicates that reduced TRbeta1 expression and tissue hypothyroidism in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage to malignant cells.

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Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis.

Publication Date: 2010 Aug 3 PMID: 20691259
Authors: Perez-Alvarez, S. - Cuenca-Lopez, M. D. - de Mera, R. M. - Puerta, E. - Karachitos, A. - Bednarczyk, P. - Kmita, H. - Aguirre, N. - Galindo, M. F. - Jordan, J.
Journal: Biochim Biophys Acta

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X(L) and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a "bioenergetic crisis." A decreased level of cellular energy may predispose cells to necrotic-like cell death.

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Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells.

Publication Date: 2010 Aug 3 PMID: 20691249
Authors: Ibey, B. L. - Pakhomov, A. G. - Gregory, B. W. - Khorokhorina, V. A. - Roth, C. C. - Rassokhin, M. A. - Bernhard, J. A. - Wilmink, G. J. - Pakhomova, O. N.
Journal: Biochim Biophys Acta

BACKGROUND: Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-mus EP. METHODS: Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry. RESULTS: 10-ns EP caused apoptotic or necrotic death within 2-20h. Survival (S, %) followed the absorbed dose (D, J/g) as: S=alphaD((-K)), where coefficients K and alpha determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas alpha was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only. CONCLUSIONS: 1.8- and 9-mus EP cause cell death efficiently and indiscriminately (LD(50) 1-3J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD(50) 50-80J/g for Jurkat and 400-500J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms. GENERAL SIGNIFICANCE: Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

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Primary effect of 1alpha,25(OH)(2)D(3) on IL-10 expression in monocytes is short-term down-regulation.

Publication Date: 2010 Aug 4 PMID: 20691220
Authors: Matilainen, J. M. - Husso, T. - Toropainen, S. - Seuter, S. - Turunen, M. P. - Gynther, P. - Yla-Herttuala, S. - Carlberg, C. - Vaisanen, S.
Journal: Biochim Biophys Acta

The biologically most active vitamin D compound, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), influences the status of inflammation by modulating the expression of several cytokine genes. In this study, we have examined the mechanism of transcriptional regulation of interleukin 10 (IL-10) by 1alpha,25(OH)(2)D(3) in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative PCR showed that IL-10 mRNA expression was significantly down-regulated (2.8-fold) during the first 8h of 1alpha,25(OH)(2)D(3) treatment, while after 48h it was up-regulated (3-fold). Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays showed that the vitamin D receptor (VDR) binds in a cyclical fashion to a promoter region 1500-1700bp upstream of the IL-10 transcription start site (TSS) containing two conserved VDR binding sites. Targeting of VDR binding sites by enhancer specific duplex RNAs revealed that only the more distal element is functional and chromosome conformation capture analysis suggested that this region loops 1alpha,25(OH)(2)D(3)-dependently to the TSS. Quantitative ChIP and micrococcal nuclease assays also revealed 1alpha,25(OH)(2)D(3)-dependent cyclical epigenetic changes and nucleosome remodeling at this promoter region. In conclusion, in LPS-treated THP-1 cells the primary effect of 1alpha,25(OH)(2)D(3) on IL-10 expression is down-regulation, which is achieved via a cyclical recruitment of VDR to the promoter.

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Thymosin beta4 induces the expression of vascular endothelial growth factor (VEGF) in a hypoxia-inducible factor (HIF)-1alpha-dependent manner.

Publication Date: 2010 Aug 4 PMID: 20691219
Authors: Jo, J. O. - Kim, S. R. - Bae, M. K. - Kang, Y. J. - Ock, M. S. - Kleinman, H. K. - Cha, H. J.
Journal: Biochim Biophys Acta

Thymosin beta4 has multi-functional roles in cell physiology, but little is known about its mechanism(s) of action. We previously reported that thymosin beta4 stimulated angiogenesis through the induction of vascular endothelial growth factor (VEGF). To identify the mechanism of VEGF induction by thymosin beta4, we have used a luciferase assay system with VEGF in the 5' promoter region. We also analyzed the effect of thymosin beta4 on VEGF mRNA stability and on the expression and stability of hypoxia-inducible factor (HIF)-1alpha. We found that thymosin beta4 induces VEGF expression by an increase in the stability of HIF-1alpha protein. Analysis of the expression patterns of thymosin beta4 and HIF-1alpha in colon cancer tissue microarray showed that thymosin beta4 and HIF-1alpha co-localized in these biopsies. These data show that thymosin beta4 induces the expression of VEGF indirectly by increasing the protein stability of HIF-1alpha.

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A novel class of photo-triggerable liposomes containing DPPC:DC(8,9)PC as vehicles for delivery of doxorubcin to cells.

Publication Date: 2010 Aug 4 PMID: 20691151
Authors: Yavlovich, A. - Singh, A. - Blumenthal, R. - Puri, A.
Journal: Biochim Biophys Acta

Success of nanoparticle-mediated drug delivery is subject to development of optimal drug release strategies within defined space and time (triggered release). Recently, we reported a novel class of photo-triggerable liposomes prepared from dipalmitoyl phosphatidylcholine (DPPC) and photopolymerizable diacetylene phospholipid (DC(8),(9)PC), that efficiently released entrapped calcein (a water soluble fluorescent dye) upon UV (254nm) treatment. To develop these formulations for in vivo applications, we have examined phototriggering of these liposomes by visible light, and the effect of released anticancer drugs on cellular toxicity. Sonicated liposomes containing various ratios of DPPC:DC(8),(9)PC and 4mol% DSPE-PEG2000 were loaded with calcein (Ex/Em, 485/517nm) or a chemotherapy drug, Doxorubicin (DOX, Ex/Em 490/590nm). Our initial experiments showed that 514nm laser treatment of liposomes containing 10 or 20mol% DC(8,9)PC for 1-3min resulted in significant release of calcein. Based on these results, we performed studies with DOX-loaded liposomes. First, biophysical properties (including liposome size and stability) and DOX encapsulation efficiency of the liposomes were determined. Subsequently, the effect of 514nm laser on DOX release, and cellular toxicity by released DOX were examined. Since liposomes using the 86:10:04 mole ratio of DPPC:DC(8),(9)PC:DSPE-PEG2000, showed highest encapsulation of DOX, these formulations were investigated further. We report that (i) liposomes retained about 70% of entrapped DOX at 37 degrees C in the presence of 0-50% serum. (ii) 514nm laser treatment resulted in DOX release from liposomes in a wavelength-specific manner. (iii) Laser treatment of co-cultures containing DOX-loaded liposomes and cells (Raji and MCF-7) resulted in at least 2-3 fold improved cell killing as compared to untreated samples. Taken together, the photo-triggerable liposomes described here may provide a platform for future drug delivery applications. To our knowledge, this is the first report demonstrating improved cell killing following light-triggered release of an encapsulated anticancer agent from photosensitive liposomes.

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Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2).

Publication Date: 2010 Aug 4 PMID: 20691150
Authors: Edwards, N. - Anderson, C. M. - Gatfield, K. M. - Jevons, M. P. - Ganapathy, V. - Thwaites, D. T.
Journal: Biochim Biophys Acta

The H(+)-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[(3)H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC(50) 1.6+/-0.4mM), alpha-methyl-d,l-tryptophan (3.5+/-1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor alpha-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and alpha-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.

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The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes.

Publication Date: 2010 Aug 4 PMID: 20691149
Authors: Dezi, M. - Fribourg, P. F. - Di Cicco, A. - Arnaud, O. - Marco, S. - Falson, P. - Di Pietro, A. - Levy, D.
Journal: Biochim Biophys Acta

ABCG2 is a human membrane ATP-binding cassette half-transporter that hydrolyzes ATP to efflux a large number of chemotherapeutic agents. Several oligomeric states of ABCG2 from homodimers to dodecamers have been reported depending on the overexpression systems and/or the protocols used for purification. Here, we compared the oligomeric state of His(6)-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His(6)-ABCG2 was purified through a new approach involving its specific recognition onto a functionalized lipid layer containing a Ni-NTA lipid. This approach allowed the purification of His-ABCG2 in presence of all solubilized membrane components that might be involved in the stabilisation of native oligomers and without requiring any additional washing or concentration passages. ABCG2 purified onto the NiNTA lipid surfaces were directly analyzed by electron microscopy and by biochemical assays. Altogether, our data are consistent with a tetrameric organization of ABCG2 when expressed in either heterologous Sf9 insect cells or in human homologous cells.

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Mitoxantrone is expelled by the ABCG2 multidrug transporter directly from the plasma membrane.

Publication Date: 2010 Aug 4 PMID: 20691148
Authors: Homolya, L. - Orban, T. I. - Csanady, L. - Sarkadi, B.
Journal: Biochim Biophys Acta

ABC multidrug transporter proteins expel a wide variety of structurally unrelated, mostly hydrophobic compounds from cells. The special role of these transporters both at the physiological barriers and in cancer cells is based on their extremely broad substrate recognition. Since hydrophobic compounds are known to partition into the lipid bilayer and accumulate in membranes, the "classical pump" model for the mechanism of multidrug transporter proteins has been challenged, and alternative models suggesting substrate recognition within the lipid bilayer have been proposed. Although much effort has been made to validate this concept, unambiguous evidence for direct drug extrusion from the plasma membrane has not been provided yet. Here we show a detailed on-line microscopic analysis of cellular extrusion of fluorescent anti-cancer drugs, mitoxantrone and pheophorbide A, by a key human multidrug transporter, ABCG2. Using the fully active GFP-tagged ABCG2 and exploiting the special character of mitoxantrone that gains fluorescence in the lipid environment, we were able to determine transporter-modulated drug concentrations separately in the plasma membrane and the intracellular compartments. Different kinetic models describing the various transport mechanisms were generated and the experimental data were analyzed using these models. On the basis of the kinetic analysis, drug extrusion from the cytoplasm can be excluded, thus, our results indicate that ABCG2 extrudes mitoxantrone directly from the plasma membrane.

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Ice-induced partial unfolding and aggregation of an integral membrane protein.

Publication Date: 2010 Aug 4 PMID: 20691147
Authors: Garber Cohen, I. P. - Castello, P. R. - Flecha, F. L.
Journal: Biochim Biophys Acta

Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253K, with t((1/2))=3.9+/-0.6h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca(2+)-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2+/-0.3 (fast cooling) and 3.7+/-0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.

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Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase.

Publication Date: 2010 Aug 4 PMID: 20691146
Authors: Marcia, M. - Langer, J. D. - Parcej, D. - Vogel, V. - Peng, G. - Michel, H.
Journal: Biochim Biophys Acta

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.

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Proton transport coupled ATP synthesis by the purified yeast H(+)-ATP synthase in proteoliposomes.

Publication Date: 2010 Aug 4 PMID: 20691145
Authors: Forster, K. - Turina, P. - Drepper, F. - Haehnel, W. - Fischer, S. - Graber, P. - Petersen, J.
Journal: Biochim Biophys Acta

The H(+)/ATP synthase from yeast mitochondria, MF(0)F(1), was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF(0)F(1) liposomes were energized by acid-base transitions (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s(-1) at a transmembrane pH difference of 3.2 units (at pH(in)=4.8 and pH(out)=8.0) and a Deltaphi of 133mV (Nernst potential). Functional studies showed that the monomeric MF(0)F(1) was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species H(2)PO(4)(-).

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Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding.

Publication Date: 2010 Sep PMID: 20682463
Authors: Chevigne, A. - Dumez, M. E. - Dumoulin, M. - Matagne, A. - Jacquet, A. - Galleni, M.
Journal: Biochim Biophys Acta

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. RESULTS: We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. GENERAL SIGNIFICANCE: Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.

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Role of ubiquitin-proteasome-mediated proteolysis in nervous system disease.

Publication Date: 2010 Aug 3 PMID: 20674814
Authors: Hegde, A. N. - Upadhya, S. C.
Journal: Biochim Biophys Acta

Proteolysis by the ubiquitin-proteasome pathway (UPP) is now widely recognized as a molecular mechanism controlling myriad normal functions in the nervous system. Also, this pathway is intimately linked to many diseases and disorders of the brain. Among the diseases connected to the UPP are neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. Perturbation in the UPP is also believed to play a causative role in mental disorders such as Angelman syndrome. The pathology of neurodegenerative diseases is characterized by abnormal deposition of insoluble protein aggregates or inclusion bodies within neurons. The ubiquitinated protein aggregates are believed to result from dysfunction of the UPP or from structural changes in the protein substrates which prevent their recognition and degradation by the UPP. An early effect of abnormal UPP in diseases of the nervous system is likely to be impairment of synaptic function. Here we discuss the UPP and its physiological roles in the nervous system and how alterations in the UPP relate to development of nervous system diseases.

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cAMP-mediated regulation of HNF-4alpha depends on the level of coactivator PGC-1alpha.

Publication Date: 2010 Sep PMID: 20670916
Authors: Dankel, S. N. - Hoang, T. - Flageng, M. H. - Sagen, J. V. - Mellgren, G.
Journal: Biochim Biophys Acta

Hepatocyte nuclear factor-4 alpha (HNF-4alpha) is a member of the nuclear receptor superfamily with important roles in hepatic metabolism. Fasting induces the cAMP/protein kinase A (PKA)-signaling pathway. The mechanisms whereby cAMP regulates HNF-4alpha transcriptional activity are incompletely understood. We have therefore investigated the role of cAMP/PKA in regulation of HNF-4alpha in COS-1 cells and the hepatoma HepG2 cell line. cAMP/PKA inhibited the transcriptional activity of HNF-4alpha in COS-1 cells, whereas a stimulatory effect was observed in HepG2 cells. The cAMP-induced inhibition of HNF-4alpha in COS-1 cells was counteracted by overexpression of the nuclear receptor coactivator PGC-1alpha, and cAMP/PKA-dependent induction of the PGC1A gene in HepG2 cells seems to explain the cell specific differences. This was further supported by knock-down of PGC-1alpha in HepG2 cells, which abolished the stimulatory effect of PKA on HNF-4alpha transcriptional activity. Similar to the cAMP/PKA-mediated regulation of HNF-4alpha, overexpression of the cAMP-response element binding protein (CREB) inhibited the transcriptional activity of HNF-4alpha in COS-1 cells, regardless of cAMP/PKA activation and CREB phosphorylation. Moreover, activation of CREB by cAMP/PKA further stimulated HNF-4alpha transactivation in HepG2 cells. cAMP induced the expression of the HNF-4alpha target genes PCK1 and G6Pase in these cells. In conclusion, our results suggest that the level of PGC-1alpha determines whether the cAMP/PKA-pathway overall stimulates or inhibits HNF-4alpha transcriptional activation.

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The levels of both lipid rafts and raft-located acetylcholinesterase dimers increase in muscle of mice with muscular dystrophy by merosin deficiency.

Publication Date: 2010 Sep PMID: 20670915
Authors: Moral-Naranjo, M. T. - Montenegro, M. F. - Munoz-Delgado, E. - Campoy, F. J. - Vidal, C. J.
Journal: Biochim Biophys Acta

Wild type and dystrophic (merosin-deficient) Lama2dy mice muscles were compared for their density of lipid rafts. The 5-fold higher level of caveolin-3 and the 2-3 times higher level of ecto-5'-nucleotidase activity in raft preparations (Triton X-100-resistant membranes) of dystrophic muscle supported expansion of caveolar and non-caveolar lipid rafts. The presence in rafts of glycosylphosphatidylinositol (GPI)-linked acetylcholinesterase (AChE) dimers, which did not arise from erythrocyte or nerve, not only revealed for the first time the capacity of the myofibre for translating the AChE-H mRNA but also an unrecognized pathway for targeting AChE-H to specialized membrane domains of the sarcolemma. Rafts of dystrophic muscle contained a 5-fold higher AChE activity/mg protein. RT-PCR for 3'-alternative mRNAs of AChE revealed AChE-T mRNA prevailing over AChE-R and AChE-H mRNAs in wild type mouse muscle. It also displayed principal 5'-alternative AChE mRNAs with exons E1c and E1e (the latter coding for N-terminally extended subunits) and fewer with E1d, E1a and E1b. The levels of AChE and butyrylcholinesterase mRNAs were unaffected by dystrophy. Finally, the decreased level of proline-rich membrane anchor (PRiMA) mRNA in Lama2dy muscle provided for a rational explanation to the loss of PRiMA-bearing AChE tetramers in dystrophic muscle.

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DNA-binding and transcriptional activities of human HSF4 containing mutations that associate with congenital and age-related cataracts.

Publication Date: 2010 Sep PMID: 20670914
Authors: Enoki, Y. - Mukoda, Y. - Furutani, C. - Sakurai, H.
Journal: Biochim Biophys Acta

Heat shock transcription factor HSF4 is necessary for ocular lens development and fiber cell differentiation. Mutations of the human HSF4 gene have been implicated in congenital and age-related cataracts. Here, we show that HSF4 activates transcription of genes encoding crystallins and beaded filament structural proteins in lens epithelial cells. Five missense mutations that have been associated with congenital cataract inhibited DNA-binding of HSF4, which demonstrates the relationship between HSF4 mutations, loss of lens protein gene expression, and cataractogenesis. However, two missense mutations that have been associated with age-related cataract did not or only slightly alter HSF4 activity, implying that other genetic and environmental factors affect the functions of these mutant proteins.

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Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation.

Publication Date: 2010 Oct PMID: 20654742
Authors: Mehrotra, S. - Mylarappa, B. N. - Iyengar, P. - Balaram, H.
Journal: Biochim Biophys Acta

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg(2+) to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K(m) values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k(cat) value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.

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Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily.

Publication Date: 2010 Oct PMID: 20654741
Authors: Linsky, T. - Fast, W.
Journal: Biochim Biophys Acta

The pentein superfamily is a mechanistically diverse superfamily encompassing both noncatalytic proteins and enzymes that catalyze hydrolase, dihydrolase and amidinotransfer reactions on guanidine substrates. Despite generally low sequence identity, they possess a conserved structural fold and display common mechanistic themes in catalysis. The structurally characterized catalytic penteins possess a conserved core of residues that include a Cys, His and two polar, guanidine-binding residues. All known catalytic penteins use the core Cys to attack the substrate's guanidine moiety to form a covalent thiouronium adduct and all cleave one or more of the guanidine C-N bonds. The mechanistic information compiled to date supports the hypothesis that this superfamily may have evolved divergently from a catalytically promiscuous ancestor.

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Sodium or potassium efflux ATPase A fungal, bryophyte, and protozoal ATPase.

Publication Date: 2010 Oct PMID: 20650263
Authors: Rodriguez-Navarro, A. - Benito, B.
Journal: Biochim Biophys Acta

The K(+) and Na(+) concentrations in living cells are strictly regulated at almost constant concentrations, high for K(+) and low for Na(+). Because these concentrations correspond to influx-efflux steady states, K(+) and Na(+) effluxes and the transporters involved play a central role in the physiology of cells, especially in environments with high Na(+) concentrations where a high Na(+) influx may be the rule. In eukaryotic cells two P-type ATPases are crucial in these homeostatic processes, the Na,K-ATPase of animal cells and the H(+)-ATPase of fungi and plants. In fungi, a third P-type ATPase, the ENA ATPase, was discovered nineteen years ago. Although for many years it was considered to be exclusively a fungal enzyme, it is now known to be present in bryophytes and protozoa. Structurally, the ENA (from exitus natru: exit of sodium) ATPase is very similar to the sarco/endoplasmic reticulum Ca(2+) (SERCA) ATPase, and it probably exchanges Na(+) (or K(+)) for H(+). The same exchange is mediated by Na(+) (or K(+))/H(+) antiporters. However, in eukaryotic cells these antiporters are electroneutral and their function depends on a DeltapH across the plasma membrane. Therefore, the current notion is that the ENA ATPase is necessary at high external pH values, where the antiporters cannot mediate uphill Na(+) efflux. This occurs in some fungal environments and at some points of protozoa parasitic cycles, which makes the ENA ATPase a possible target for controlling fungal and protozoan parasites. Another technological application of the ENA ATPase is the improvement of salt tolerance in flowering plants.

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Evidence that the translocon may function as a hydropathy partitioning filter.

Publication Date: 2010 Oct PMID: 20646997
Authors: Mulvihill, C. M. - Deber, C. M.
Journal: Biochim Biophys Acta

Developing a greater understanding of the function of the translocon-and the source of its selectivity for transmembrane helix insertion-are important steps toward deciphering the role of disease-causing mutations in membrane regions. To address these phenomena, we have prepared a library of helix-loop-helix ("hairpin") constructs derived from helices 3 and 4 of the first membrane domain of CFTR, in which position 232 was mutated individually to each of the 20 commonly-occurring amino acids. Using retention times on a reverse phase-HPLC C18 column to mimic the process of hairpin partitioning, we have quantitatively determined a hydropathy scale in the context of a bona fide membrane protein fragment that correlates to an in vivo hydropathy scale with r=-0.78-a value that rises to r=-0.92 when Asp and Glu are excluded due to protonation effects. Our results provide evidence that the translocon may act as a facilitator in the insertion selection process, effectively allowing the bilayer to "decide" through favorable non-polar solvation whether or not to allow a translocating helix to enter the membrane.

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Monitoring mitochondrial electron fluxes using NAD(P)H-flavoprotein fluorometry reveals complex action of isoflurane on cardiomyocytes.

Publication Date: 2010 Oct PMID: 20646994
Authors: Sedlic, F. - Pravdic, D. - Hirata, N. - Mio, Y. - Sepac, A. - Camara, A. K. - Wakatsuki, T. - Bosnjak, Z. J. - Bienengraeber, M.
Journal: Biochim Biophys Acta

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes.

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ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F(1)-ATPase are affected by inorganic phosphate.

Publication Date: 2010 Oct PMID: 20646992
Authors: Milgrom, Y. M.
Journal: Biochim Biophys Acta

The effect of inorganic phosphate (P(i)) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F(1)-ATPase from beef heart mitochondria (ndMF(1)) has been investigated. It is shown for the first time that P(i) decreases the apparent rate constant of uni-site ATP binding by ndMF(1) 3-fold with the K(d) of 0.38+/-0.14mM. During uni-site ATP hydrolysis, P(i) also shifts equilibrium between bound ATP and ADP+P(i) in the direction of ATP synthesis with the K(d) of 0.17+/-0.03mM. However, 10mM P(i) does not significantly affect ATP binding during multi-site catalysis.

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Endothelial progenitor cells express PAF receptor and respond to PAF via Ca(2+)-dependent signaling.

Publication Date: 2010 Oct PMID: 20637897
Authors: Balestrieri, M. L. - Giovane, A. - Milone, L. - Servillo, L.
Journal: Biochim Biophys Acta

Endothelial progenitor cell (EPC) therapy is a promising approach to promote angiogenesis and endothelial repair in patients with cardiovascular diseases (CVD). However, their release of proinflammatory mediators may compromise the therapeutic efficacy. Little is known about the role of Platelet-Activating Factor (PAF) in EPC functional response. Here, we investigated the expression of PAF receptor (PAF-R) in early EPC and the release of PAF under stimulation with factors involved in endothelial dysfunction. Results indicated that early EPC express the PAF-R and respond to PAF signaling via a transient increase of cytoplasmic Ca(2+) concentration. EPC release PAF in a time dependent manner upon stimulation with tumor necrosis factor-alpha (TNF-alpha) or high-glucose concentration with a peak at 30min and 10min (p<0.01 vs. control), respectively. PAF, starting at concentration of 50ng/ml, exerted a detrimental effect on EPC number with a concomitant increase of p38 activity. Furthermore, both the reduction of early EPC number and the enhanced p38 activity induced by PAF were abolished by CV3988, a PAF receptor antagonist. These novel findings, revealing that early EPC respond to PAF signaling, unveil an inflammatory pathway that may play a crucial role in the outcome of cardiovascular cell therapy with EPC.

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Absence of ataxin-3 leads to cytoskeletal disorganization and increased cell death.

Publication Date: 2010 Oct PMID: 20637808
Authors: Rodrigues, A. J. - do Carmo Costa, M. - Silva, T. L. - Ferreira, D. - Bajanca, F. - Logarinho, E. - Maciel, P.
Journal: Biochim Biophys Acta

Ataxin-3 (ATXN3) is a widely expressed protein that binds to ubiquitylated proteins, has deubiquitylating activity in vitro and is thought to modulate substrate degradation through the ubiquitin-proteasome pathway. Expansion of a polyglutamine tract in ATXN3 causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation and specific neuronal death. Although ATXN3 has been involved in transcriptional repression and in the ubiquitin-proteasome pathway, its biological function is still unknown. In this work, we show that depletion of ATXN3 using small-interference RNA (siRNA) causes a prominent phenotype in both human and mouse cell lines. A mild increase in ubiquitylation occurs and cells exhibit ubiquitin-positive foci, which is consistent with ATXN3 putative function as a deubiquitylating enzyme. In addition, siATXN3-silenced cells exhibit marked morphological changes such as rounder shape and loss of adhesion protrusions. At a structural level, the microtubule, microfilament and intermediate filament networks are severely compromised and disorganized. This cytoskeletal phenotype is reversible and dependent on ATXN3 levels. Cell-extracellular matrix connection is also affected in ATXN3-depleted cells as talin expression is reduced in the focal adhesions and lower levels of alpha-1 integrin subunit are expressed at their surface. Although the cytoskeletal and adhesion problems do not originate any major change in the cell cycle of siATXN3-depleted cells, cell death is increased in siATXN3 cultures compared to controls. In summary, in this work we show that the absence of ATXN3 leads to an overt cytoskeletal/adhesion defect raising the possibility that this protein may play a role in the cytoskeleton.

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Structural contributions to the intracellular targeting strategies of antimicrobial peptides.

Publication Date: 2010 Oct PMID: 20637722
Authors: Lan, Y. - Ye, Y. - Kozlowska, J. - Lam, J. K. - Drake, A. F. - Mason, A. J.
Journal: Biochim Biophys Acta

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide-DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic alpha-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an alpha-helix structure is not required for the DNA binding and condensation activity of buforin II amide.

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Sphingomyelin analogs with branched N-acyl chains: The position of branching dramatically affects acyl chain order and sterol interactions in bilayer membranes.

Publication Date: 2010 Oct PMID: 20637720
Authors: Jaikishan, S. - Bjorkbom, A. - Slotte, J. P.
Journal: Biochim Biophys Acta

Sphingolipids have been found to have single methyl branchings both in their long-chain base and in their N-linked acyl chains. In this study we determined how methyl-branching in the N-linked acyl chain of sphingomyelin (SM) affected their membrane properties. SM analogs with a single methyl-branching at carbon 15 (of a 17:0 acyl chain; anteiso) had a lower gel-liquid transition temperature as compared to an iso-branched SM analog. Phytanoyl SM (methyls at carbons 3, 7, 11 and 15) as well as a SM analog with a methyl on carbon 10 in a hexadecanoyl chain failed to show a gel-liquid transition above 10 degrees C. Only the two distally branched SM analogs (iso and anteiso) formed ordered domains with cholesterol in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. However, domains formed by the branched SM analogs appeared to contain less sterol when compared to palmitoyl SM (PSM) as the saturated phospholipid. Sterol-enriched domains formed by the anteiso SM analog were also less stable against temperature than domains formed by PSM. Both the 10-methyl and phytanoyl SM analogs failed to form sterol-enriched domains in the POPC bilayer. Acyl chain branching weakened SM/sterol interactions markedly when compared to PSM, as also evidenced from the decreased affinity of cholestatrienol to bilayers containing branched SM analogs. Our results show that methyl-branching weakened intermolecular interactions in a position-dependent manner.

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Vacuolar (H(+))-ATPases in Caenorhabditis elegans: What can we learn about giant H(+) pumps from tiny worms?

Publication Date: 2010 Oct PMID: 20637717
Authors: Lee, S. K. - Li, W. - Ryu, S. E. - Rhim, T. - Ahnn, J.
Journal: Biochim Biophys Acta

Vacuolar (H(+))-ATPases, also called V-ATPases, are ATP-driven proton pumps that are highly phylogenetically conserved. Early biochemical and cell biological studies have revealed many details of the molecular mechanism of proton pumping and of the structure of the multi-subunit membrane complex, including the stoichiometry of subunit composition. In addition, yeast and mouse genetics have broadened our understanding of the physiological consequences of defective vacuolar acidification and its related disease etiologies. Recently, phenotypic investigation of V-ATPase mutants in Caenorhabditis elegans has revealed unexpected new roles of V-ATPases in both cellular function and early development. In this review, we discuss the functions of the V-ATPases discovered in C. elegans.

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DNA binding domain of RFX5: Interactions with X-box DNA and RFXANK.

Publication Date: 2010 Oct PMID: 20637319
Authors: Chakraborty, M. - Sengupta, A. - Bhattacharya, D. - Banerjee, S. - Chakrabarti, A.
Journal: Biochim Biophys Acta

Regulatory factor X (RFX) is a heterotrimeric protein complex having RFX5, RFXANK and RFXAP as its three subunits. It is involved in the regulation of the transcription of MHCII molecules in antigen presenting cells. The RFX complex binds to X-box DNA, using the DNA binding domain, present in RFX5. The DNA binding domain (DBD) of RFX5 (12kD) and intact RFXANK (35kD) were subcloned, expressed and purified. The associations of RFX5DBD with the X-box DNA and between RFX5DBD and RFXANK were measured in this study. The interaction of RFX5DBD and X-box DNA was studied using steady state fluorescence quenching and circular dichroism. The binding dissociation constant (K(d)) of the DNA-protein complex was determined from fluorescence measurements. The van't Hoff plot was linear over the temperature range 10-25 degrees C and the binding was found to be entropy-driven and enthalpy-favorable. The effect of electrolytes in RFX5DBD-DNA association was also studied. Molecular association between RFX5DBD and RFXANK has been observed by fluorescence resonance energy transfer (FRET) measurements, changes in the ratio of the two vibronic intensities of pyrene labeled RFX5DBD in presence of RFXANK and chemical cross-linking followed by tandem mass spectrometry. Results showed that the two proteins could interact in the absence of the third subunit RFXAP, in vitro with an apparent dissociation constant (K(d)) of 128nM.

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Isolation of short peptide fragments from alpha-synuclein fibril core identifies a residue important for fibril nucleation: A possible implication for diagnostic applications.

Publication Date: 2010 Oct PMID: 20637318
Authors: Yagi, H. - Takeuchi, H. - Ogawa, S. - Ito, N. - Sakane, I. - Hongo, K. - Mizobata, T. - Goto, Y. - Kawata, Y.
Journal: Biochim Biophys Acta

alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinson's disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.

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Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.

Publication Date: 2010 Oct PMID: 20637315
Authors: Ribeiro, T. - Santos-Silva, T. - Alves, V. D. - Dias, F. M. - Luis, A. S. - Prates, J. A. - Ferreira, L. M. - Romao, M. J. - Fontes, C. M.
Journal: Biochim Biophys Acta

Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as alpha, beta and gamma. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the gamma sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of beta and gamma sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities.

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A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state.

Publication Date: 2010 Oct PMID: 20637181
Authors: Tolleter, D. - Hincha, D. K. - Macherel, D.
Journal: Biochim Biophys Acta

Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic alpha-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.

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S-resistin inhibits adipocyte differentiation and increases TNFalpha expression and secretion in 3T3-L1 cells.

Publication Date: 2010 Oct PMID: 20627112
Authors: Fernandez, C. M. - Del Arco, A. - Gallardo, N. - Aguado, L. - Rodriguez, M. - Ros, M. - Carrascosa, J. M. - Andres, A. - Arribas, C.
Journal: Biochim Biophys Acta

S-resistin is a non-secretable resistin spliced variant described in white adipose tissue from Wistar rats. Since resistin has been implicated in adipogenesis regulation, here we have investigated the possible role of this new isoform in this process. For that, we have studied the adipocyte development in 3T3-L1 pre-adipocyte cell line stably expressing s-resistin and resistin. Both isoforms are able to restrain 3T3-L1 pre-adipocyte differentiation though affecting differently the expression pattern of pro-adipogenic transcription factors such CCAAT/enhancer binding proteins alpha and beta (C/EBPalpha and C/EBPbeta) and peroxisome proliferator-activated receptor gamma (PPARgamma), as well of proteins implicated in lipid metabolism such perilipin, fatty acid synthase (FAS), adipocyte lipid binding protein (ALBP/aP2) and carnitine palmitoyltransferase1 (CPT1). Likewise, both resistin isoforms impair insulin-stimulated glucose transport by decreasing glucose transport 4 (GLUT4) expression but to a different degree. In addition, s-resistin expressing 3T3-L1 cells display other remarkable differences. Thus, in these cells, endogenous resistin expression falls down while tumor necrosis factor alpha (TNFalpha) and interleukine 6 (IL-6) productions are increased along differentiation. These findings indicate that s-resistin isoform also impairs adipocyte differentiation affecting the expression pattern of key pro-adipogenic transcription factors and insulin sensitivity. Additionally, s-resistin may play a role in inflammatory processes.

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Identification of differentially expressed transcripts and translatants targeted by knock-down of endogenous PCBP1.

Publication Date: 2010 Oct PMID: 20624489
Authors: Huo, L. R. - Ju, W. - Yan, M. - Zou, J. H. - Yan, W. - He, B. - Zhao, X. L. - Jenkins, E. C. - Brown, W. T. - Zhong, N.
Journal: Biochim Biophys Acta

PCBP1 is a member of the hnRNP family and participates in the regulation of transcription and translation. Previously, we identified transcripts targeted by overexpression of exogenous PCBP1. To further determine if these altered transcripts may also be targeted by a lack of PCBP1, we depleted endogenous PCBP1 in human SH-SY5Y cells. We identified 941 transcripts with the Affymetrix and 1362 with the Agilent expression platforms. There were 375 transcripts identified by both platforms, including 328 down-regulated and 47 up-regulated. The identified transcripts could be grouped into neuronal, cell signaling, metabolic, developmental, and differentiation categories, with pathway involvement in Wnt signaling, TGF beta signaling, translation factors and nuclear receptors. A proteomic profiling study with a two-dimensional chromatographic platform showed global translational changes over a range of isoelectric points (pI)=4.84-8.42. This study identifies the transcripts affected by knock-down of endogenous PCBP1 and compares them to the transcripts affected by overexpression of PCBP1.

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Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A(4) hydrolase.

Publication Date: 2010 Oct PMID: 20624488
Authors: Seipelt, R. L. - Bailey, F. C. - Schaible, A. - Thompson, M. W.
Journal: Biochim Biophys Acta

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A(4) hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.

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Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis.

Publication Date: 2010 Oct PMID: 20624480
Authors: Pereira, M. - Song, Z. - Santos-Silva, L. K. - Richards, M. H. - Nguyen, T. T. - Liu, J. - de Almeida Soares, C. M. - da Silva Cruz, A. H. - Ganapathy, K. - Nes, W. D.
Journal: Biochim Biophys Acta

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a K(mapp) of 38muM and k(catapp) of 0.14min(-1). Quite unexpectedly, "plant" cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-(13)C]- or [24-(2)H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of (1)H and (13)CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Delta(24(25))-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K(i) 14nM) and 26,27-dehydrolanosterol (K(i) 54muM and k(inact) of 0.24min(-1)) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC(50), 30nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C(1)-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.

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Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes.

Publication Date: 2010 Sep PMID: 20621583
Authors: Porteous, C. M. - Logan, A. - Evans, C. - Ledgerwood, E. C. - Menon, D. K. - Aigbirhio, F. - Smith, R. A. - Murphy, M. P.
Journal: Biochim Biophys Acta

BACKGROUND: Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential. CONCLUSIONS: Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function. METHODS: To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration. RESULTS: AlkylTPP cations were accumulated selectively by mitochondria within mice within 5min of iv injection. The extent of uptake was enhanced 10-30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration. GENERAL SIGNIFICANCE: These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.

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The glycosylation of myeloperoxidase.

Publication Date: 2010 Oct PMID: 20621206
Authors: Ravnsborg, T. - Houen, G. - Hojrup, P.
Journal: Biochim Biophys Acta

The enzyme myeloperoxidase (MPO) is an important part of the neutrophil immune reaction and can be found in alfa granula. The presence of MPO can be used to distinguish acute myelogenous leukemia from acute lymphocytic leukemia. However, the methods employed to do so, such as flow cytometry and immunohistochemistry rely on antibody recognition, and therefore the characterization of the mature MPO, including post-translational modifications, must be considered as important as epitope mapping. MPO has 5 N-linked glycosylation sites, occupied by both high mannose and complex glycan structures. In this study we utilize intact glycopeptide MSMS analysis for site specific characterization of the glycan structures of MPO from a cancer patient. The identified glycan structures are compared to those of MPO from healthy donors, in order to probe for any potential differences that may have diagnostic use.

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A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

Publication Date: 2010 Oct PMID: 20621130
Authors: Peltonen, H. M. - Akerman, K. E. - Bart, G.
Journal: Biochim Biophys Acta

The neuropeptides orexin-A/hypocretin-1 (Ox-A) and orexin-B/hypocretin-2 play an important role in the control of energy metabolism via either of two G-protein-coupled receptors, orexin receptor 1 (Ox1R) and 2. Despite its significant physiological functions, signaling via orexin receptors is still poorly characterized. The aim of this study was to improve our understanding of early signaling events triggered by the binding of Ox-A to Ox1R. Using phosphospecific antibodies, we observed that early kinase activation by Ox-A in a HEK293 cell line stably expressing Ox1R (HEKOx1R) included ERK1/2, PKCdelta, and PKD1. Elevation of intracellular Ca(2+) is a well-characterized response to Ox1R activation. Comparison of Ox-A-induced calcium elevation and PKD1 activation demonstrated that both responses are detectable soon after stimulation and increase in a dose-dependent manner, but inhibition of protein kinase C, when low Ox-A concentrations are used, affects them differently. PKD family of protein kinases has 3 members: PKD1, 2, and 3, which are all expressed in HEKOx1R cells. In response to stimulation of the cells with 1nM Ox-A, both PKD1 and PKD3 are activated and increased in the plasma membrane, pointing at a possible role for these kinases in that cell compartment. Overexpression of either kinase-dead PKD1 or kinase-dead PKD3 disrupts Ox-A-induced calcium oscillations demonstrating the functional role of these kinases in modulating physiological responses to Ox-A.

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Histone deacetylase 7 (HDAC7) regulates myocyte migration and differentiation.

Publication Date: 2010 Oct PMID: 20621129
Authors: Gao, C. - Liu, Y. - Lam, M. - Kao, H. Y.
Journal: Biochim Biophys Acta

Class IIa HDACs including HDAC7 play a role in gene expression, cell differentiation, and animal development through their association with transcription factors such as myogenic enhancer factors 2 (MEF2s). In this study, we show that endogenous HDAC7 localizes to both the nucleus and the cytoplasm of C2C12 myoblasts but is exclusively retained in the cytoplasm of myotubes after completion of differentiation process. To elucidate the role of differential distribution of HDAC7 during myogenesis, we examined the effects of stably expressed HDAC7 mutants on myogenesis. Expression of nuclear-retained HDAC7 mutants significantly inhibits myogenesis in C2C12 cells and reduces the expression of muscle-specific myosin heavy chain (MHC) and myogenin. The inhibition in myocyte differentiation can be partially relieved by introduction of a mutation disrupting HDAC7:MEF2 interaction. Since phosphorylation of HDAC7 plays an important role in its nucleocytoplasmic shuttling, we further investigated the expression and distribution of phosphorylated HDAC7. To our surprise, the phosphorylation levels of HDAC7 at S344 and S479 were slightly decreased upon differentiation, whereas the phosphorylation of S178 was unchanged. Interestingly, a significant fraction of pS344- and/or pS479-HDAC7 localized to plasma membrane of myotubes. In addition, Ser178-phosphorylated (pS178) HDAC7 displays a predominately actin filament-like structure before muscle differentiation. Consistent with this notion, HDAC7 partially colocalized with actin filaments; in particular, pS178-HDAC7 largely colocalized with actin filaments as indicated by phalloidin counter staining in myocytes. Furthermore, C2C12 cells expressing nuclear-retained HDAC7 display defects in migration. Our results provide novel insight into the mechanisms that regulate myocyte differentiation and migration by controlling the subcellular distribution of HDAC7 in differentiating myoblasts.

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3-D modelling of chloroplast structure under (Mg(2+)) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking.

Publication Date: 2010 Oct PMID: 20621057
Authors: Rumak, I. - Gieczewska, K. - Kierdaszuk, B. - Gruszecki, W. I. - Mostowska, A. - Mazur, R. - Garstka, M.
Journal: Biochim Biophys Acta

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.

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Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I.

Publication Date: 2010 Oct PMID: 20620244
Authors: Manokaran, S. - Banerjee, J. - Mallik, S. - Srivastava, D. K.
Journal: Biochim Biophys Acta

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15A deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme-ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn(2+) cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2kcal/mol (DeltaDeltaG composite function) and 0.89kcal/mol (DeltaDeltaG(double dagger)) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.

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Non-mammalian fat-1 gene prevents neoplasia when introduced to a mouse hepatocarcinogenesis model Omega-3 fatty acids prevent liver neoplasia.

Publication Date: 2010 Oct PMID: 20620224
Authors: Griffitts, J. - Saunders, D. - Tesiram, Y. A. - Reid, G. E. - Salih, A. - Liu, S. - Lydic, T. A. - Busik, J. V. - Kang, J. X. - Towner, R. A.
Journal: Biochim Biophys Acta

We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-alpha (transforming growth factor-alpha), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFalpha/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFalpha/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-kappaB (nuclear factor-kappaB) levels at 40weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.

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An Abeta concatemer with altered aggregation propensities.

Publication Date: 2010 Oct PMID: 20619363
Authors: Giehm, L. - Dal Degan, F. - Fraser, P. - Klysner, S. - Otzen, D. E.
Journal: Biochim Biophys Acta

We present an analysis of the conformational and aggregative properties of an Abeta concatemer (Con-Alz) of interest for vaccine development against Alzheimer's disease. Con-Alz consists of 3 copies of the 43 residues of the Abeta peptide separated by the P2 and P30 T-cell epitopes from the tetanus toxin. Even in the presence of high concentrations of denaturants or fluorinated alcohols, Con-Alz has a very high propensity to form aggregates which slowly coalesce over time with changes in secondary, tertiary and quaternary structure. Only micellar concentrations of SDS were able to inhibit aggregation. The increase in the ability to bind the fibril-binding dye ThT increases without lag time, which is characteristic of relatively amorphous aggregates. Confirming this, electron microscopy reveals that Con-Alz adopts a morphology resembling truncated protofibrils after prolonged incubation, but it is unable to assemble into classical amyloid fibrils. Despite its high propensity to aggregate, Con-Alz does not show any significant ability to permeabilize vesicles, which for fibrillating proteins is taken to be a key factor in aggregate cytotoxicity and is attributed to oligomers formed at an early stage in the fibrillation process. Physically linking multiple copies of the Abeta-peptide may thus sterically restrict Con-Alz against forming cytotoxic oligomers, forcing it instead to adopt a less well-organized assembly of intermeshed polypeptide chains.

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Medium osmolarity-dependent biosynthesis of renal cellular sulfoglycolipids is mediated by the MAPK signaling pathway.

Publication Date: 2010 Oct PMID: 20619354
Authors: Niimura, Y. - Moue, T. - Takahashi, N. - Nagai, K. I.
Journal: Biochim Biophys Acta

Verots S3 and Vero317 cells were shown by metabolic labeling with (35)S-sulfate to contain many more sulfoglycosphingolipids than original Vero cells derived from African green monkey kidney. The activity of galactosyl ceramide sulfotransferase (GST) was shown to be 89- and 92-fold higher in Vero317 cells and Verots S3 cells, respectively, than that of the parent cells, whereas the activity of the degradation enzyme, arylsulfatase A, was unchanged among all the three cell strains. GST gene transcript levels in Verots cells were 14.3-fold higher than those in Vero cells. The cell adhesiveness to the culture plate under hypertonic stress was strengthened significantly in both mutant strains. Among the major sulfoglycolipids of the Verots S3 cell line, assigned as SM4s, SM3, SM2a, and SB1a, the incorporation of (35)S-sulfate into SM3, SM2a and SB1a was upregulated with the increasing tonicity of the medium. Sulfoglycolipids in these renal cells seemed to contribute to the membrane barrier against hypertonic media as shown previously in another renal cell line, MDCK (Niimura and Nagai, 2008). Sulfoglycolipid synthesis was suppressed with the p38 (MAPK) inhibitor SB203580 and/or with the MEK-1/2 (MAPKK) inhibitor PD98059, and with the tyrosine kinase inhibitor genistein, which also reduced the sulfoglycolipid synthesis in a dose-dependent manner. Further the administration of the MAPK/MAPKK inhibitors to the culture medium reduced significantly the viability of Verots S3 cells under hypertonic stress. These findings suggest that sulfoglycolipid synthesis in those renal cells may be regulated to adapt to the renal osmotic circumstances by the medium's osmolarity via the MAPK signaling pathway.

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Mild hyperoxia limits hTR levels, telomerase activity, and telomere length maintenance in hTERT-transduced bone marrow endothelial cells.

Publication Date: 2010 Oct PMID: 20619302
Authors: Napier, C. E. - Veas, L. A. - Kan, C. Y. - Taylor, L. M. - Yuan, J. - Wen, V. W. - James, A. - O'Brien, T. A. - Lock, R. B. - Mackenzie, K. L.
Journal: Biochim Biophys Acta

Reactivation of telomerase in endothelial cells (ECs) may be an effective approach to the treatment of vascular disorders associated with telomere attrition and EC senescence. However, overexpression of human telomerase reverse transcriptase (hTERT) does not prevent net telomere loss in ECs grown in standard culture medium with exposure to atmospheric oxygen (21% O(2)). Since these culture conditions are hyperoxic relative to normal tissue in vivo, where oxygen tension is estimated to be 1%-6%, we examined the effects of reduced exposure to oxidative stress (OS) on telomere length maintenance in hTERT-transduced bone marrow endothelial (BMhTERT) cells. Propagation of BMhTERT cells in the free radical scavenger, tert-butylhydroxylamine (tBN), and/or in 5% O(2) increased telomerase enzyme activity and facilitated telomere length maintenance. The enhancement of telomerase activity correlated with higher levels of the telomerase RNA component (hTR). We also investigated the role of the telomere binding protein, TRF1, in telomere length regulation under alternate OS conditions. Inhibition of TRF1 function had no effect on telomere length in BMhTERT cells grown under standard culture conditions. However, alleviation of OS by growth in tBN plus 5% O(2), elevated hTR levels, enhanced telomerase enzyme activity, and enabled progressive telomere lengthening. The direct impact of hTR levels on telomerase-mediated telomere lengthening was demonstrated by overexpression of hTR. BMhTERT cells transduced with hTR exhibited very high telomerase enzyme activity and underwent dramatic telomere lengthening under standard culture conditions. Overall, these results demonstrate that hTR levels are reduced by mild hyperoxia and limit telomerase-mediated telomere lengthening in hTERT-transduced ECs.

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Folding and unfolding characteristics of short beta strand peptides under different environmental conditions and starting configurations.

Publication Date: 2010 Oct PMID: 20615483
Authors: Maass, A. - Tekin, E. D. - Schuller, A. - Palazoglu, A. - Reith, D. - Faller, R.
Journal: Biochim Biophys Acta

We analyze the effect of different environmental conditions, sequence lengths and starting configurations on the folding and unfolding pathways of small peptides exhibiting beta turns. We use chignolin and a sequence of peptide G as examples. A variety of different analysis tools allows us to characterize the changes in the folding pathways. It is observed that different harmonic modes dominate not only for different conditions but also for different starting points. The modes remain essentially very similar but their relative importance varies. A detailed analysis from diverse viewpoints including the influence of the particular amino acid sequence, conformational aspects as well as the associated motions yields a global picture that is consistent with experimental evidence and theoretical studies published elsewhere. Patterns of modes that remain stable over a range of temperatures might serve as an additional diagnostic to identify conformations that have reliably adopted a native fold. This could aid in reconstructing the folding process of a complete protein by identifying conformationally determined regions.

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The membrane environment modulates self-association of the human GpA TM domain-Implications for membrane protein folding and transmembrane signaling.

Publication Date: 2010 Oct PMID: 20603102
Authors: Anbazhagan, V. - Schneider, D.
Journal: Biochim Biophys Acta

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.

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Alzheimer's disease amyloid-beta peptide analogue alters the ps-dynamics of phospholipid membranes.

Publication Date: 2010 Oct PMID: 20603101
Authors: Buchsteiner, A. - Haubeta, T. - Dante, S. - Dencher, N. A.
Journal: Biochim Biophys Acta

We have investigated the influence of the neurotoxic Alzheimer's disease peptide amyloid-beta (25-35) on the dynamics of phospholipid membranes by means of quasi-elastic neutron scattering in the picosecond time-scale. Samples of pure phospholipids (DMPC/DMPS) and samples with amyloid-beta (25-35) peptide included have been compared. With two different orientations of the samples the directional dependence of the dynamics was probed. The sample temperature was varied between 290K and 320K to cover both the gel phase and the liquid-crystalline phase of the lipid membranes. The model for describing the dynamics combines a long-range translational diffusion of the lipid molecules and a spatially restricted diffusive motion. Amyloid-beta (25-35) peptide affects significantly the ps-dynamics of oriented lipid membranes in different ways. It accelerates the lateral diffusion especially in the liquid-crystalline phase. This is very important for all kinds of protein-protein interactions which are enabled and strongly influenced by the lateral diffusion such as signal and energy transducing cascades. Amyloid-beta (25-35) peptide also increases the local lipid mobility as probed by variations of the vibrational motions with a larger effect in the out-of-plane direction. Thus, the insertion of amyloid-beta (25-35) peptide changes not only the structure of phospholipid membranes as previously demonstrated by us employing neutron diffraction (disordering effect on the mosaicity of the lipid bilayer system) but also the dynamics inside the membranes. The amyloid-beta (25-35) peptide induced membrane alteration even at only 3mol% might be involved in the pathology of Alzheimer's disease as well as be a clue in early diagnosis and therapy.

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Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1.

Publication Date: 2010 Oct PMID: 20603100
Authors: Kim, J. K. - Lee, S. A. - Shin, S. - Lee, J. Y. - Jeong, K. W. - Nan, Y. H. - Park, Y. S. - Shin, S. Y. - Kim, Y.
Journal: Biochim Biophys Acta

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro(8) for Gly(8) in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly(8) of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro(8) whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys(8). Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].

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Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers.

Publication Date: 2010 Oct PMID: 20603098
Authors: Orzechowska, A. - Lipinska, M. - Fiedor, J. - Chumakov, A. - Zajac, M. - Slezak, T. - Matlak, K. - Strzalka, K. - Korecki, J. - Fiedor, L. - Burda, K.
Journal: Biochim Biophys Acta

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mossbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~165K) than that of the low spin atom (~207K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17meV) and higher (17-25meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~6meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q(A) and Q(B)) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.

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In rat dipeptidyl peptidase III, His(568) is essential for catalysis, and Glu(507) or Glu(512) stabilizes the coordination bond between His(455) or His(450) and zinc ion.

Publication Date: 2010 Oct PMID: 20601226
Authors: Fukasawa, K. M. - Hirose, J. - Hata, T. - Ono, Y.
Journal: Biochim Biophys Acta

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, "HELLGH," as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K(m) values or in the substrate specificity. However, the k(cat) values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His(568) residue of rat DPP III is essential for enzyme catalysis. The k(cat) values of the mutants E507A and E512A were 2.38 and 3.88s(-1) toward Arg-Arg-NA, and 0.097 and 0.59s(-)(1) toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5x10(3)to2x10(4). The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu(507) and Glu(512) form the hydrogen bonds to the nitrogen atoms of His(455) and His(450). All of these results showed that Glu(507) or Glu(512) stabilizes the coordination bond between the zinc ion and His(455) or His(450).

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Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite.

Publication Date: 2010 Oct PMID: 20601225
Authors: Bonaventura, C. - Henkens, R. - De Jesus-Bonilla, W. - Lopez-Garriga, J. - Jia, Y. - Alayash, A. I. - Siburt, C. J. - Crumbliss, A. L.
Journal: Biochim Biophys Acta

The clam Lucina pectinalis supports its symbiotic bacteria by H(2)S transport in the open and accessible heme pocket of Lucina Hb I and by O(2) transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O(2) was removed from its active site. Structural analysis revealed that O(2) "clams up" the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O(2) affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O(2). These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins.

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Propofol lowers serum PF4 level and partially corrects hypercoagulopathy in endotoxemic rats.

Publication Date: 2010 Sep PMID: 20601223
Authors: Tang, J. - Sun, Y. - Wu, W. K. - Zhong, T. - Liu, Y. - Xiao, J. - Tao, T. - Zhao, Z. - Gu, M.
Journal: Biochim Biophys Acta

Propofol, an anesthetic drug, has been shown to exhibit antioxidant and anti-inflammatory properties in vitro and in vivo. Hypercoagulopathy is a common clinical feature of sepsis, but the effects of propofol on the coagulation system in septic conditions are unclear. Using the gel-based comparative proteomic approach, together with Western blot analysis, ELISA, antithrombin III activity assay, and blood coagulation test, the effect of propofol on serum proteomic profiles in endotoxemic rats was examined. We identified that serum platelet factor-4 (PF4), an endogenous pro-coagulant, was up-regulated in LPS-challenged rats (p<0.001). Endotoxemia also resulted in hypercoagulopathy as evidenced by the shortening of thromboplastin time and thrombin time. Administration of propofol attenuated LPS-stimulated PF4 release and partially reversed the effect of LPS on thromboplastin time (p=0.0012) and thrombin time (p=0.0072). We demonstrated that propofol reduces serum levels of PF4 and partially corrects the hypercoagulopathy associated with endotoxemia in rats.

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Analysis of binding modes of ligands to multiple conformations of CYP3A4.

Publication Date: 2010 Oct PMID: 20601222
Authors: Teixeira, V. H. - Ribeiro, V. - Martel, P. J.
Journal: Biochim Biophys Acta

Cytochromes P450 (CYPs) are extremely versatile enzymes capable of catalyzing a vast number of compounds, and CYP3A4 is no exception metabolizing approximately half of the currently marketed drugs, besides endogenous compounds. To metabolize such a variety of compounds, CYP3A4 has to be extremely flexible, which makes interaction studies difficult. We employ a multi-conformational docking setup where conformations are generated by several molecular dynamics simulations to analyze the binding modes of various ligands, and the docking is considered successful if the ligand site of catalysis (SOC) is within 6.0A of the haem Fe. While docking with the X-ray structure proved unsuccessful with all ligands, the multi-conformational docking achieved successful binding of each ligand to at least one protein conformation. Analysis of the docked solutions highlights residues in the active site cavity that may have an important role in access, binding and stabilization of the ligand.

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An interpretation of positional displacement of the helix12 in nuclear receptors: Preexistent swing-up motion triggered by ligand binding.

Publication Date: 2010 Sep PMID: 20601221
Authors: Watanabe, C. - Watanabe, H. - Tanaka, S.
Journal: Biochim Biophys Acta

Positional displacement of helix12 (H12) in the estrogen receptor alpha, which belongs to the nuclear receptor (NR) superfamily, is studied by the molecular dynamics (MD) simulation and the linear response theory. Tendency of the H12 to swing up upon ligand binding, which is consistent with X-ray structures and earlier MD simulations, is reproduced by the calculation of the conformational fluctuation in apo state and the response to the external perturbation. Our study thus provides an interpretation of the positional change of the H12 such that it is derived by the preexistent swing-up motion where the ligand binding works only as a trigger. Our finding, which illustrates underlying mechanism of the H12 motion, would contribute to finding a way to regulate the transcriptional activity by synthesized ligands because the transcriptional activity of the NR is governed by the position of the H12.

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Reversible binding of zinc in Plasmodium falciparum Sir2: Structure and activity of the apoenzyme.

Publication Date: 2010 Sep PMID: 20601220
Authors: Chakrabarty, S. P. - Balaram, H.
Journal: Biochim Biophys Acta

Reversible zinc chelation via thiol groups of cysteines leading to modulation of activity in redox regulated proteins forms a basis for switching on-off of various biochemical processes. Silent information regulator 2 (Sir2), a NAD(+) dependent deacetylase, contains a non-catalytic zinc ion coordinated by thiol groups of cysteines. Using Plasmodium falciparum Sir2 (PfSir2), we have examined the effect of zinc removal on the structure and activity of this enzyme. Our studies show that the enzyme with high affinity for zinc exhibits partial collapse of structure upon removal of the metal ion. Zinc reconstitution of apo PfSir2 led to recovery of both structure and activity highlighting the reversibility of the process.

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Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme-substrate docking studies.

Publication Date: 2010 Sep PMID: 20601218
Authors: Jung, J. - Park, H. J. - Uhm, K. N. - Kim, D. - Kim, H. K.
Journal: Biochim Biophys Acta

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.

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Endogenous H(2)O(2) produced by Streptococcus pneumoniae controls FabF activity.

Publication Date: 2010 Sep PMID: 20601114
Authors: Benisty, R. - Cohen, A. Y. - Feldman, A. - Cohen, Z. - Porat, N.
Journal: Biochim Biophys Acta

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H(2)O(2). Streptococcus pneumoniae produces exceptionally high levels of H(2)O(2), mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H(2)O(2) inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H(2)O(2) production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H(2)O(2). In vitro exposure of the spxB mutant to various H(2)O(2) concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H(2)O(2) or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H(2)O(2) produced by S. pneumoniae.

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Expression and role of Elovl4 elongases in biosynthesis of very long-chain fatty acids during zebrafish Danio rerio early embryonic development.

Publication Date: 2010 Oct PMID: 20601113
Authors: Monroig, O. - Rotllant, J. - Cerda-Reverter, J. M. - Dick, J. R. - Figueras, A. - Tocher, D. R.
Journal: Biochim Biophys Acta

Elovl4 is a fatty acyl elongase that participates in the biosynthesis of very long-chain fatty acids (>/=C24), which are relatively abundant in skin (saturated chains), or retina, brain and testes (polyunsaturated chains) of mammals. In the present study we characterised two Elovl4 proteins, Elovl4a and Elovl4b, from zebrafish Danio rerio, and investigated their expression patterns during embryonic development. Heterologous expression in baker's yeast showed that both zebrafish Elovl4 proteins efficiently elongated saturated fatty acids up to C36, with 26:0 appearing the preferred substrate as reported for human ELOVL4. Interestingly, activity for the elongation of PUFA substrates was only shown by Elovl4b, which effectively converted eicosapentaenoic (20:5n-3) and arachidonic (20:4n-6) acids to elongated polyenoic products up to C36. Furthermore, zebrafish Elovl4b may be involved in the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) as it had the capacity to elongate 22:5n-3 to 24:5n-3 which can be subsequently desaturated and chain shortened to DHA in peroxisomes. The distinct functional roles of zebrafish Elovl4 proteins were also reflected in their spatial-temporal expression patterns during ontogeny. Analyses by whole-mount in situ hybridisation in zebrafish embryos showed that elovl4a was expressed in neuronal tissues (wide-spread distribution in the head area), with elovl4b specifically expressed in epiphysis (pineal gland) and photoreceptor cells in the retina. Similarly, tissue distribution in adults revealed that elovl4a transcripts were found in most tissues analysed, whereas elovl4b expression was essentially restricted to eye and gonads. Overall, the results suggest that zebrafish elovl4b resembles other mammalian orthologues in terms of function and expression patterns, whereas elovl4a may represent an alternative elongase not previously described in vertebrates.

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Docosahexaenoic acid and eicosapentaenoic acid are converted by 3T3-L1 adipocytes to N-acyl ethanolamines with anti-inflammatory properties.

Publication Date: 2010 Oct PMID: 20601112
Authors: Balvers, M. G. - Verhoeckx, K. C. - Plastina, P. - Wortelboer, H. M. - Meijerink, J. - Witkamp, R. F.
Journal: Biochim Biophys Acta

n-3 PUFAs have beneficial health effects which are believed to be partly related to their anti-inflammatory properties, however the exact mechanisms behind this are unknown. One possible explanation could be via their conversion to N-acyl ethanolamines (NAEs), which are known to possess anti-inflammatory properties. Using fatty acid precursors we showed that 3T3-L1 adipocytes are indeed able to convert docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to their NAE derivatives docosahexaenoyl ethanolamine (DHEA) and eicosapentaenoyl ethanolamine (EPEA), respectively. This synthesis took place on top of an apparent background formation of these NAEs in standard culture medium. In addition we were able to demonstrate the presence of DHEA, but not of EPEA, in human plasma. DHEA and EPEA were found to decrease LPS induced adipocyte IL-6 and MCP-1 levels. Results of combined incubations with PPAR-gamma and CB2 antagonists suggest a role of these receptors in mediating the reduction of IL-6 by DHEA. Our results are in line with the hypothesis that in addition to other pathways, formation of N-acyl ethanolamines may contribute to the biological activity of n-3 PUFAs. Different targets, including the endocannabinoid system, may be involved in the immune-modulating activity of these "fish-oil-derived NAEs."

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Direct evidence for leptin-induced lipid oxidation independent of long-form leptin receptor.

Publication Date: 2010 Oct PMID: 20601111
Authors: Akasaka, Y. - Tsunoda, M. - Ogata, T. - Ide, T. - Murakami, K.
Journal: Biochim Biophys Acta

Leptin administration has been shown to enhance muscle lipid oxidation in relation to the energy expenditure. Both long-form (Ob-R(L)) and short-form leptin receptors (Ob-R(S)) are expressed in skeletal muscle, but the role of Ob-R(S) is unclear. In the present study, the role of Ob-R(S) in leptin-induced lipid oxidation in skeletal muscles was investigated using primary murine myotubes from m/m and db/db mice. Primary myotubes were treated with leptin (0.1, 1, 10, 100nM) for 24h. Lipid oxidation was determined by (14)CO(2) production rate from [1-(14)C] palmitate. Leptin was found to increase lipid oxidation in a dose- and time-dependent manner in db/db myotubes as well as in m/m myotubes. Leptin significantly increased phosphorylation of JAK2 and STAT3 in both types of myotube. Leptin-induced lipid oxidation was abolished by STAT3 siRNA. To investigate the mechanism underlying leptin-induced lipid oxidation, the effects of pharmacological inhibitors were examined. JAK2 or p38 MAPK inhibitor suppressed leptin-induced lipid oxidation and decreased STAT3 phosphorylation in both types of myotube, respectively. Leptin significantly increased phosphorylation of p38 MAPK, and leptin-induced lipid oxidation was abolished by treatment with p38 MAPK siRNA in both types of myotube. These results suggest that leptin induces lipid oxidation in skeletal muscle through the JAK2/p38 MAPK/STAT3 signaling pathway via not only Ob-R(L) but also Ob-R(S).

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Molecular characterization of dopamine-derived quinones reactivity toward NADH and glutathione: Implications for mitochondrial dysfunction in Parkinson disease.

Publication Date: 2010 Sep PMID: 20600874
Authors: Bisaglia, M. - Soriano, M. E. - Arduini, I. - Mammi, S. - Bubacco, L.
Journal: Biochim Biophys Acta

Oxidative stress and mitochondrial dysfunction, especially at the level of complex I of the electronic transport chain, have been proposed to be involved in the pathogenesis of Parkinson disease (PD). A plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (DA) and produce various toxic molecules, i.e., free radicals and quinone species (DAQ). It has been shown that DA oxidation products can induce various forms of mitochondrial dysfunction, such as mitochondrial swelling and decreased electron transport chain activity. In the present work, we analyzed the potentially toxic effects of DAQ on mitochondria and, specifically, on the NADH and GSH pools. Our results demonstrate that the generation of DAQ in isolated respiring mitochondria triggers the opening of the permeability transition pore most probably by inducing oxidation of NADH, while GSH levels are not affected. We then characterized in vitro, by UV and NMR spectroscopy, the reactivity of different DA-derived quinones, i.e., dopamine-o-quinone (DQ), aminochrome (AC) and indole-quinone (IQ), toward NADH and GSH. Our results indicate a very diverse reactivity for the different DAQ studied that may contribute to unravel the complex molecular mechanisms underlying oxidative stress and mitochondria dysfunction in the context of PD.

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Selective detection of Cathepsin E proteolytic activity.

Publication Date: 2010 Sep PMID: 20600629
Authors: Abd-Elgaliel, W. R. - Tung, C. H.
Journal: Biochim Biophys Acta

BACKGROUND: Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide-substrate that is only sensitive to Cath E but not Cath D. METHODS: Substrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH(2), designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence. RESULTS: Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (K(cat)/K(M)) of Cath E for substrate e was 16.7muM(-)(1)S(-)(1). No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B. CONCLUSIONS: This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement. GENERAL SIGNIFICANCE: This study forms the foundation of Cath E specific inhibitor development in further studies.

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Proliferative and anti-proliferative effects of retinoic acid at doses similar to endogenous levels in Leydig MLTC-1/R2C/TM-3 cells.

Publication Date: 2010 Sep PMID: 20600628
Authors: Perri, M. - Pingitore, A. - Cione, E. - Vilardi, E. - Perrone, V. - Genchi, G.
Journal: Biochim Biophys Acta

BACKGROUND: Vitamin A is suggested to be protective against oxidative stress. However, different authors observed pro-oxidant effects of retinoids both in experimental works and clinical trials. These discordances are the bases for the investigation of the proliferative and anti-proliferative properties of retinoic acid (RA) in biological systems. METHODS: Cell viability is determined with the MTT assay. Oxidative stress parameters are detected measuring catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. FABP5 mRNA levels are measured by RT-PCR. Autophagy and apoptosis are analyzed by Monodansylcadaverine (MDC) staining and TUNEL assay, respectively. RESULTS AND CONCLUSIONS: RA, at nutraceutic/endogenous doses (10-200nM), increases cell viability of testes tumor Leydig cell lines (MLTC-1 and R2C) and modulates antioxidant enzyme activities, as CAT and GST. RA is able to induce proliferation through non-classical and redox-dependent mechanisms accompanied by increased levels of FABP5 mRNA. The redox environment of the cell is currently thought to be extremely important for controlling either apoptosis or autophagy. Apoptosis occurs at pharmacological doses, while autophagy, which plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis, is triggered at the critical concentration of 500nM RA, both in normal and tumoral cells. Slight variations of RA concentrations are evaluated as a threshold value to distinguish between the proliferative or anti-proliferative effects. GENERAL SIGNIFICANCE: Although retinoids have a promising role as antineoplastic agents, physiological levels of RA play a key role in Leydig cancer progression, fostering proliferation and growth of testicular tumoral mass.

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A monovalent agonist of TrkA tyrosine kinase receptors can be converted into a bivalent antagonist.

Publication Date: 2010 Sep PMID: 20600627
Authors: Brahimi, F. - Liu, J. - Malakhov, A. - Chowdhury, S. - Purisima, E. O. - Ivanisevic, L. - Caron, A. - Burgess, K. - Saragovi, H. U.
Journal: Biochim Biophys Acta

BACKGROUND: Receptor tyrosine kinases (RTK) act through dimerization. Previously it was thought that only bivalent ligands could be agonistic, whereas monovalent ligands should be antagonistic. This notion changed after the demonstration that monovalent ligands can be agonistic, including our report of a small molecule monovalent ligand "D3" that is a partial agonist of the NGF receptor TrkA. A bivalent "D3-linker-D3" was expected to increase agonism. METHODS: Dimeric analogs were synthesized and tested in binding, biochemical, and biological assays. RESULTS: One analog, 1-ss, binds TrkA with higher affinity than D3 and induces or stabilizes receptor dimers. However, 1-ss exhibited antagonistic activity, through two mechanisms. One mechanism is that 1-ss blocks NGF binding, unlike D3 which is non-competitive. Inhibition of NGF binding may be due to the linker of 1-ss filling the inter-receptor space that NGF traverses before docking. In a second mechanism, 1-ss acts as a pure antagonist, inhibiting NGF-independent TrkA activity in cells over-expressing receptors. Inhibition is likely due to 1-ss "freezing" the TrkA dimer in the inactive state. CONCLUSIONS: Dimerization of an RTK can result in antagonism, through two independent mechanisms. GENERAL SIGNIFICANCE: we report a small molecule monovalent agonist being converted to a bivalent antagonist.

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Cloning and functional characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the methylerythritol phosphate pathway for isoprenoid biosynthesis.

Publication Date: 2010 Sep PMID: 20600626
Authors: Perez-Gil, J. - Bergua, M. - Boronat, A. - Imperial, S.
Journal: Biochim Biophys Acta

BACKGROUND: The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans. RESULTS: This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli. GENERAL SIGNIFICANCE: Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities. CONCLUSION: The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.

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Telomeric DNA-binding activities of heterogeneous nuclear ribonucleoprotein A3 in vitro and in vivo.

Publication Date: 2010 Oct PMID: 20600361
Authors: Huang, P. R. - Hung, S. C. - Wang, T. C.
Journal: Biochim Biophys Acta

Telomeres are dynamic DNA-protein complexes that protect the ends of linear chromosome. Telomere-binding proteins play crucial role in the maintenance of telomeres. HnRNP A3 has been shown recently to bind specifically to single-stranded telomeric DNA in vitro, although its in vivo telomere function remains unknown. In this study, the DNA-binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. The minimal sequence for hnRNP A3 binding to DNA was determined as an undecamer with the following consensus sequence 5'-[T/C]AG[G/T]NN[T/C]AG[G/T]N-3'. Confocal microscopy and chromatin-immunoprecipitation (ChIP) analyses showed that hnRNP A3 is associated with telomere in vivo. Knocking-down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation. In contrast, overexpression of hnRNP A3 resulted in the production of steady-state short telomeres in OECM1 cells. These results suggest that hnRNP A3 is associated with telomere in vivo and acts as a negative regulator of telomere length maintenance.

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Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots.

Publication Date: 2010 Oct PMID: 20600360
Authors: Heidbreder, M. - Endesfelder, U. - van de Linde, S. - Hennig, S. - Widera, D. - Kaltschmidt, B. - Kaltschmidt, C. - Heilemann, M.
Journal: Biochim Biophys Acta

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

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The evolution of protein targeting and translocation systems.

Publication Date: 2010 Oct PMID: 20600359
Authors: Bohnsack, M. T. - Schleiff, E.
Journal: Biochim Biophys Acta

Cells have evolved increasingly complex membrane systems for compartmentalization and thereby for the regulation of multiple cellular pathways. The existence of such membranes required the evolution of molecular machines that allow and regulate the exchange of material between intracellular compartments or with the exterior. Here, we have summarized the current concepts for the origin and evolution of the targeting and translocation systems required for the specific insertion of transmembrane proteins into their target membranes and for the transport of protein cargos across membranes. The basic pathways developed in prokaryotes were modified and extended to suffice for the much more complex membrane systems found in eukaryotes, allowing not only the identification of basic mechanistic principles, but also phylogenetic studies to elucidate evolutionary relations.

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Lipid rafts modulate the activation but not the maintenance of store-operated Ca(2+) entry.

Publication Date: 2010 Sep PMID: 20600358
Authors: Galan, C. - Woodard, G. E. - Dionisio, N. - Salido, G. M. - Rosado, J. A.
Journal: Biochim Biophys Acta

Different studies have reported that proteins involved in Ca(2+) entry are localized in discrete plasma membrane domains known as lipid rafts, which have been suggested to support store-operated Ca(2+) entry by facilitating STIM1 clustering in endoplasmic reticulum-plasma membrane junctions as well as the interaction of STIM1 with TRPC1. Here we report that treatment of HEK293 cells with thapsigargin (TG) results in the activation of Ca(2+) entry with two components, an early, La(3+)-sensitive, component and a late component that shows both La(3+)-sensitive and -insensitive constituents. Preincubation with methyl-beta-cyclodextrin (MbetaCD) prevented TG-induced activation of Ca(2+) entry but, in contrast, enhanced this process after its activation. Addition of MbetaCD after store depletion did not modify the La(3+)-sensitive store-operated divalent cation entry but increased La(3+)-insensitive non-capacitative Ca(2+) entry. Cell stimulation with TG results in a transient increase in Orai1 co-immunoprecipitation with STIM1, TRPC1 and TRPC6. TG-induced association of these proteins was significantly attenuated by preincubation for 30min with MbetaCD, without altering surface expression of Orai1 or TRPCs. In contrast, the association of Orai1 with STIM1 or TRPC1 was unaffected when MbetaCD was added after store depletion with TG. Addition of MbetaCD to TG-treated cells promoted dissociation between Orai1 and TRPC6, as well as non-capacitative Ca(2+) entry. TRPC6 expression silencing indicates that MbetaCD-enhanced non-capacitative Ca(2+) entry was mediated by TRPC6. In conclusion, lipid raft domains are necessary for the activation but not the maintenance of SOCE probably due to the support of the formation of Ca(2+) signalling complexes involving Orai1, TRPCs and STIM1.

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Galectin-3: A novel substrate for c-Abl kinase.

Publication Date: 2010 Oct PMID: 20600357
Authors: Balan, V. - Nangia-Makker, P. - Jung, Y. S. - Wang, Y. - Raz, A.
Journal: Biochim Biophys Acta

Galectin-3, a ss-galactoside-binding lectin, is found in cellular and extracellular location of the cell and has pleiotropic biological functions such as cell growth, cell adhesion and cell-cell interaction. It may exhibit anti- or pro-apoptotic activity depending on its localization and post-translational modifications. Two important post-translational modifications of galectin-3 have been reported: its cleavage and phosphorylation. Cleavage of galectin-3 was reported to be involved with angiogenic potential and apoptotic resistance. Phosphorylation of galectin-3 regulates its sugar-binding ability. In this report we have identified novel tyrosine phosphorylation sites in galectin-3 as well as the kinase responsible for its phosphorylation. Our results demonstrate that tyrosines at positions 79, 107 and 118 can be phosphorylated in vitro and in vivo by c-Abl kinase. Tyrosine 107 is the main target of c-Abl. Expression of galectin-3 Y107F mutant in galectin-3 null SK-Br-3 cells leads to morphological changes and increased motility compared to wild type galectin-3. Further investigation is needed to better understand the functional significance of the novel tyrosine phosphorylated sites of galectin-3.

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Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775.

Publication Date: 2010 Oct PMID: 20599694
Authors: Joshi, S. - Bisht, G. S. - Rawat, D. S. - Kumar, A. - Kumar, R. - Maiti, S. - Pasha, S.
Journal: Biochim Biophys Acta

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 mug/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.

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Comparative 5-doxylstearoyllecithin and 3-doxylcholestane EPR spin labeling study of phospholipid bilayer perturbation by different oxidized lecithin species.

Publication Date: 2010 Oct PMID: 20599693
Authors: Megli, F. M. - Conte, E. - Russo, L.
Journal: Biochim Biophys Acta

A 3-doxylcholestane spin label was employed in addition to 5-doxylstearoyl lecithin for a more detailed study of the different effects exerted by variously oxidized lecithins on fatty acid alignment in phospholipid planar bilayers. Either spin label was enclosed in oriented PLPC planar samples also containing in turn a variety of conjugated-dienes lecithins and cleaved chain lecithins, in order to monitor EPR spectral angular dependence loss. Data obtained by use of arachidonoyl-hydroxystearoyl-PC and palmitoyl-hydroxylinoleoyl-PC confirm that the 5-DSPC nitroxide ring almost completely retains its orientation in CD-PCs-containing planar membranes, in contrast with angular dependence loss observed in the presence of the CC-PC molecular species palmitoyl-oxononanoyl-PC and palmitoyl-oxovaleroyl-PC, already seen with cleaved-chain palmitoyl-glutaroyl-PC and palmitoyl-azelaoyl-PC. However, the 3-DC nitroxide ring also loses its orientation with CD-PCs, in addition to being disoriented by cleaved chain-lecithins, similarly to 5DSPC. Joint information from the two spin labels will help to clarify whether OXPC-related disordering involved the whole bilayer structure or only the hydrophobic core. In addition, the propensity of different OXPCs to form bilayer vesicles in water suspension was also determined by Sepharose 4B gel-chromatography. The results suggest that CD-PCs might yield SPB bilayer structures with a disordered hydrophobic core, while pure CC-PC more probably forms non-bilayer disordered structures, possibly micelles or mixed micelle/bilayers.

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Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography.

Publication Date: 2010 Oct PMID: 20599691
Authors: Trepout, S. - Taveau, J. C. - Benabdelhak, H. - Granier, T. - Ducruix, A. - Frangakis, A. S. - Lambert, O.
Journal: Biochim Biophys Acta

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonasaeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.

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Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis.

Publication Date: 2010 Oct PMID: 20599687
Authors: Lee, T. H. - Heng, C. - Swann, M. J. - Gehman, J. D. - Separovic, F. - Aguilar, M. I.
Journal: Biochim Biophys Acta

Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5muM), indicating a surface-associated state without significant perturbation in membrane structure. At 10muM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20muM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action.

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YTPdb: A wiki database of yeast membrane transporters.

Publication Date: 2010 Oct PMID: 20599686
Authors: Brohee, S. - Barriot, R. - Moreau, Y. - Andre, B.
Journal: Biochim Biophys Acta

Membrane transporters constitute one of the largest functional categories of proteins in all organisms. In the yeast Saccharomyces cerevisiae, this represents about 300 proteins ( approximately 5% of the proteome). We here present the Yeast Transport Protein database (YTPdb), a user-friendly collaborative resource dedicated to the precise classification and annotation of yeast transporters. YTPdb exploits an evolution of the MediaWiki web engine used for popular collaborative databases like Wikipedia, allowing every registered user to edit the data in a user-friendly manner. Proteins in YTPdb are classified on the basis of functional criteria such as subcellular location or their substrate compounds. These classifications are hierarchical, allowing queries to be performed at various levels, from highly specific (e.g. ammonium as a substrate or the vacuole as a location) to broader (e.g. cation as a substrate or inner membranes as location). Other resources accessible for each transporter via YTPdb include post-translational modifications, K(m) values, a permanently updated bibliography, and a hierarchical classification into families. The YTPdb concept can be extrapolated to other organisms and could even be applied for other functional categories of proteins. YTPdb is accessible at http://homes.esat.kuleuven.be/ytpdb/.

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Determinants of membrane association in the SH4 domain of Fyn: Roles of N-terminus myristoylation and side-chain thioacylation.

Publication Date: 2010 Oct PMID: 20599685
Authors: Rawat, A. - Nagaraj, R.
Journal: Biochim Biophys Acta

The SH4 domain of Fyn, a member of the Src family of tyrosine kinases, though rich in polar amino acid residues, anchors to the cytosolic face of membranes upon fatty acylation. In order to probe the requirement of specific fatty acylation at the N-terminus and at the side-chain of this domain for membrane-association, we have studied the interaction of peptides corresponding to the polar segment of the SH4 domain of Fyn and its mono- and dually fatty acylated analogs with model membranes. While the polar segment without covalently linked fatty acids (KDKEATKLTEW-amide) does not interact with lipid vesicles, peptides with one covalently linked fatty acid at the N-terminus or in the side-chain, associate with zwitterionic and anionic lipids to varying degrees. The interaction of dually acylated peptides (Myr-GK(epsilon-myr)KDKEATKLTEW-amide and Myr-GC(S-pal)KDKEATKLTEW-amide) with lipids depends on the linkage between fatty acyl side-chain and peptide backbone. The peptide chain associates with membranes only when the side-chain acylation is via an amide bond and not via a thioester bond. Our investigations indicate that acylation is essential for membrane targeting and unacylated polar stretch of the SH4 domain does not have a role in membrane-anchoring. Side-chain acylation via a thioester bond not only provides membrane anchorage but also directs the peptide chain away from the bilayer which might be important to enable the full length protein to interact with other signaling partners.

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The plasma membrane plays a central role in cells response to mechanical stress.

Publication Date: 2010 Sep PMID: 20599684
Authors: Verstraeten, S. V. - Mackenzie, G. G. - Oteiza, P. I.
Journal: Biochim Biophys Acta

The mechanisms by which lymphocytes recognize and interpret mechanical stimuli and translate these into the triggering of select signaling cascades that are critical for lymphocyte function are still not fully understood. In this work, we investigated the association of mechanical stress (MS)-induced changes in membrane physical properties with changes in cytoskeleton dynamics and cell signaling. In Jurkat T cells, MS was associated with the immediate and transient depolymerization of both beta-tubulin and F-actin. The fluidity of the plasma membrane measured in the hydrophobic region of the bilayer, increased 0.5min post-MS, recovering the initial value in the following 2min. This effect was accompanied by the rearrangement of lipids in the lateral phase of the plasma membrane, transient lipid rafts' alteration, and membrane hyperpolarization. The consequent increase in cellular [Ca(2+)] triggered the activation of the transcription factors NFAT, AP-1, and NF-kappaB. Results indicate that the cytoplasmic membrane, through changes in membrane physical properties, senses MS, and transduces an initial physical stimulus into microtubules rearrangements, Ca(2+) mobilization, and the subsequent changes in cell signaling.

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Interactions of oritavancin, a new semi-synthetic lipoglycopeptide, with lipids extracted from Staphylococcus aureus.

Publication Date: 2010 Oct PMID: 20599683
Authors: Domenech, O. - Dufrene, Y. F. - Van Bambeke, F. - Tukens, P. M. - Mingeot-Leclercq, M. P.
Journal: Biochim Biophys Acta

Oritavancin, a lipoglycopeptide with marked bactericidal activity against vancomycin-resistant Staphylococcus aureus and enterococci, induces calcein release from CL:POPE and POPG:POPE liposomes, an effect enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG (Domenech et al., Biochim Biophys Acta 2009; 1788:1832-40). Using vesicles prepared from lipids extracted from S. aureus, we showed that oritavancin induces holes, erosion of the edges, and decrease of the thickness of the supported lipid bilayers (atomic force microscopy; AFM). Oritavancin also induced an increase of membrane permeability (calcein release) on a time- and dose-dependent manner. These effects were probably related to the ability of the drug to bind to lipid bilayers as shown by 8-anilino-1- naphthalene sulfonic acid (ANS) assay. Interaction of oritavancin with phospholipids at the level of their glycerol backbone and hydrophobic domain was studied by monitoring changes of Laurdan excitation generalized polarization (GP(ex)) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy upon temperature increase. Oritavancin increased GP(ex) values and the transition temperature, indicating a more ordered structure at the level of the glycerol backbone. Oritavancin slightly decreased DPH fluorescence depolarization intensities, suggesting an increase in fluidity at the level of acyl chains. Together, our data confirm the interaction of oritavancin with lipids and the potential role of a rigidifying effect at the level of glycerol backbone for membrane permeabilization. This work shows how AFM and biophysical methods may help in characterizing drug-membrane interactions, and sheds further light on the mode of action of oritavancin.

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Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase.

Publication Date: 2010 Sep PMID: 20599681
Authors: Ramzan, R. - Staniek, K. - Kadenbach, B. - Vogt, S.
Journal: Biochim Biophys Acta

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (Psi(m)) in living cells and tissues at low values (100-140mV), when the matrix ATP/ADP ratios are high. In contrast, high Psi(m) values (180-220mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of Psi(m) from 233 to 123mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of Psi(m) is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of Psi(m) was found. From the data we conclude that high matrix ATP/ADP ratios keep Psi(m) at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the Psi(m)-independent "allosteric ATP-inhibition of CcO." Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by "respiratory control," based on Psi(m) according to the Mitchell Theory.

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Occurrence, biosynthesis and function of isoprenoid quinones.

Publication Date: 2010 Sep PMID: 20599680
Authors: Nowicka, B. - Kruk, J.
Journal: Biochim Biophys Acta

Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K(1), a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.

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GDP and carboxyatractylate inhibit 4-hydroxynonenal-activated proton conductance to differing degrees in mitochondria from skeletal muscle and heart.

Publication Date: 2010 Oct PMID: 20599679
Authors: Aguirre, E. - Cadenas, S.
Journal: Biochim Biophys Acta

The lipid peroxidation product 4-hydroxynonenal (HNE) increases the proton conductance of the inner mitochondrial membrane through effects on uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT); however, the relative contribution of the two carriers to these effects is unclear. To clarify this we isolated mitochondria from skeletal muscle and heart of wild-type and Ucp3 knockout (Ucp3KO) mice. To increase UCP3 expression, some mice were i.p. injected with LPS (12mg/kg body weight). In spite of the increased UCP3 expression levels, basal proton conductance did not change. HNE increased the proton conductance of skeletal muscle and heart mitochondria. In skeletal muscle, this increase was lower in Ucp3KO mice and higher in LPS-treated wild-type mice, and was partially abolished by GDP (UCPs inhibitor) and completely abolished by carboxyatractylate (ANT inhibitor) or addition of both inhibitors. GDP had no effect on HNE-induced conductance in heart mitochondria, but carboxyatractylate or administration of both inhibitors had a partial effect. GDP-mediated inhibition of HNE-activated proton conductance in skeletal muscle mitochondria was not observed in Ucp3KO mice, indicating that GDP is specific for UCP3, at least in muscle. Carboxyatractylate was able to inhibit UCP3, probably through an indirect mechanism. Our results are consistent with the conclusion that, in skeletal muscle, HNE-induced increase in proton conductance is mediated by UCP3 (30%) and ANT, whereas in the heart the increase is mediated by ANT and other carriers, possibly including UCP3.

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Triplet-triplet energy transfer in the major intrinsic light-harvesting complex of Amphidinium carterae as revealed by ODMR and EPR spectroscopies.

Publication Date: 2010 Oct PMID: 20599677
Authors: Di Valentin, M. - Salvadori, E. - Agostini, G. - Biasibetti, F. - Ceola, S. - Hiller, R. - Giacometti, G. M. - Carbonera, D.
Journal: Biochim Biophys Acta

We present an optically detected magnetic resonance (ODMR) and electron paramagnetic resonance (EPR) spectroscopic study on the quenching of photo-induced chlorophyll triplet states by carotenoids, in the intrinsic light-harvesting complex (LHC) from the dinoflagellate Amphidinium carterae. Two carotenoid triplet states, differing in terms of optical and magnetic spectroscopic properties, have been identified and assigned to peridinins located in different protein environment. The results reveal a parallelism with the triplet-triplet energy transfer (TTET) process involving chlorophyll a and luteins observed in the LHC-II complex of higher plants. Starting from the hypothesis of a conserved alignment of the amino acid sequences at the cores of the LHC and LHC-II proteins, the spin-polarized time-resolved EPR spectra of the carotenoid triplet states of LHC have been calculated by a method which exploits the conservation of the spin momentum during the TTET process. The analysis of the spectra shows that the data are compatible with a structural model of the core of LHC which assigns the photo-protective function to two central carotenoids surrounded by the majority of Chl a molecules present in the protein, as found in LHC-II. However, the lack of structural data, and the uncertainty in the pigment composition of LHC, leaves open the possibility that this complex posses a different arrangement of the pigments with specific centers of Chl triplet quenching.

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Stability curves of laboratory evolved thermostable mutants of a Bacillus subtilis lipase.

Publication Date: 2010 Sep PMID: 20599630
Authors: Kamal, M. Z. - Ahmad, S. - Yedavalli, P. - Rao, N. M.
Journal: Biochim Biophys Acta

Shape of the protein stability curves changes to achieve higher melting temperature. Broadly, these changes have been classified as upward shift (increased G(s)), rightward shift (increase in T(s)) and flattening of the stability curves (decrease in C(p)). Comparative studies on homologous mesophilic-thermophilic protein pairs highlighted the differential contribution of these three strategies amongst proteins. But unambiguous way of identification of the strategies, which will be preferred for a protein, is still not achieved. We have performed comparative thermodynamic studies using differential scanning calorimeter (DSC) on thermostable variants of a lipase from Bacillus subtilis. These variants are products of 1, 2, 3 and 4 rounds of directed evolution and harbor mutations having definite contribution in thermostability unlike natural thermophilic proteins. We have shown that upward and rightward shift in stability curves are prime strategies in this lipase. Our results along with that from the other study on laboratory evolved xylanase A suggest that optimization of suboptimal thermodynamic parameters is having a dominant influence in selection of thermodynamic strategies for higher thermostability.

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A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage.

Publication Date: 2010 Oct PMID: 20599567
Authors: Carlessi, L. - Buscemi, G. - Fontanella, E. - Delia, D.
Journal: Biochim Biophys Acta

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit.

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Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance.

Publication Date: 2010 Oct PMID: 20599534
Authors: Helmholz, H.
Journal: Biochim Biophys Acta

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3mug/mL and 43.1mug/mL against sheep and 13.5mug/mL and 8.8mug/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore((R)) technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4mug/mL to 35.4mug/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.

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The NMR solution structure of subunit G (G(61)(-)(101)) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae.

Publication Date: 2010 Oct PMID: 20599533
Authors: Rishikesan, S. - Manimekalai, M. S. - Gruber, G.
Journal: Biochim Biophys Acta

Subunit G is an essential stalk subunit of the eukaryotic proton pump V(1)V(O) ATPase. Previously the structure of the N-terminal region, G(1)(-)(59), of the 13kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G(61)(-)(101)) of subunit G of the Saccharomyces cerevisiae V(1)V(O) ATPase. The protein forms an extended alpha-helix between residues 64 and 100, whereby the first five- and the last residues of G(61)(-)(101) are flexible. The surface charge distribution of G(61)(-)(101) reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G(1)(-)(59), the presented solution structure of G(61)(-)(101) enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V(1)V(O) ATPase.

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The dynamics of mitochondrial Ca(2+) fluxes.

Publication Date: 2010 Oct PMID: 20599532
Authors: de la Fuente, S. - Montenegro, P. - Fonteriz, R. I. - Moreno, A. - Lobaton, C. D. - Montero, M. - Alvarez, J.
Journal: Biochim Biophys Acta

We have investigated the kinetics of mitochondrial Ca(2+) influx and efflux and their dependence on cytosolic [Ca(2+)] and [Na(+)] using low-Ca(2+)-affinity aequorin. The rate of Ca(2+) release from mitochondria increased linearly with mitochondrial [Ca(2+)] ([Ca(2+)](M)). Na(+)-dependent Ca(2+) release was predominant al low [Ca(2+)](M) but saturated at [Ca(2+)](M) around 400muM, while Na(+)-independent Ca(2+) release was very slow at [Ca(2+)](M) below 200muM, and then increased at higher [Ca(2+)](M), perhaps through the opening of a new pathway. Half-maximal activation of Na(+)-dependent Ca(2+) release occurred at 5-10mM [Na(+)], within the physiological range of cytosolic [Na(+)]. Ca(2+) entry rates were comparable in size to Ca(2+) exit rates at cytosolic [Ca(2+)] ([Ca(2+)](c)) below 7muM, but the rate of uptake was dramatically accelerated at higher [Ca(2+)](c). As a consequence, the presence of [Na(+)] considerably reduced the rate of [Ca(2+)](M) increase at [Ca(2+)](c) below 7muM, but its effect was hardly appreciable at 10muM [Ca(2+)](c). Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca(2+)](M) transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca(2+) buffering, and comparison of our results with data on total mitochondrial Ca(2+) fluxes indicate that the mitochondrial Ca(2+) bound/Ca(2+) free ratio is around 10- to 100-fold for most of the observed [Ca(2+)](M) range and suggest that massive phosphate precipitation can only occur when [Ca(2+)](M) reaches the millimolar range.

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Cell cycle control pathways act as conditioning factors for TK/GCV sensitivity in pancreatic cancer cells.

Publication Date: 2010 Oct PMID: 20599444
Authors: Abate-Daga, D. - Garcia-Rodriguez, L. - Sumoy, L. - Fillat, C.
Journal: Biochim Biophys Acta

The suicide system TK/GCV is an enzyme/prodrug therapy that involves the transfer of the cDNA for the herpes simplex virus thymidine kinase gene (TK) into tumor cells which then sensitizes the cells to the non-toxic antiviral drug ganciclovir. Although extensively characterized, the suicide system TK/GCV conceals the details of its mechanism of action. In order to shed some light on this issue, we conducted experiments designed to identify key features of sensitive cells, as compared to cells that displayed reduced sensitivity to TK/GCV. Cell lines displaying different degrees of sensitivity underwent apoptotic cell death upon treatment with TK/GCV. S-phase delay, however, was almost exclusively restricted to sensitive cells and was impaired in a model of treatment-induced resistance. In this model genes with differential expression associated to induced resistance were identified. Noteworthy, two cell cycle-related genes (CCNE1 and GADD45) were functionally validated as conditioners of cellular sensitivity to TK/GCV. The relevance of cell cycle control was further demonstrated by experiments showing the association of Chk1 activation with greater TK/GCV cytotoxicity. Combination treatment with Chk1 inhibitor UCN-01 induced, in sensitive cells, an antagonistic effect on TK/GCV cytotoxicity highlighting the relevance of Chk1's activity on TK/GCV mechanism of action. These results reveal the relevance of cell cycle control pathways in the cytotoxicity induced by the TK/GCV system identifying candidate genes as conditioners of TK/GCV sensitivity. Moreover it points out, for the first time at Chk1 activation as a key factor to mediate TK/GCV cytotoxicity.

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Proteomic analysis of primary porcine endothelial cells after infection by classical swine fever virus.

Publication Date: 2010 Sep PMID: 20595071
Authors: Li, S. - Qu, H. - Hao, J. - Sun, J. - Guo, H. - Guo, C. - Sun, B. - Tu, C.
Journal: Biochim Biophys Acta

Endothelial cells are the main target of classical swine fever virus during infection, and extensive hemorrhage is the most typical clinical sign of classical swine fever. To investigate the molecular mechanism of hemorrhagic pathogenesis, two-dimensional difference gel electrophoresis with fluorescent dyes (2D-DIGE) was used to analyze the proteomic profile of primary porcine umbilical vein endothelial cells (PUVECs) following CSFV infection. Of 15 protein spots with differential expression, 8 were characterized by MALDI-TOF-MS/MS in infected PUVECs at 48h p.i.: moesin, peroxiredoxin 6, stathmin-1, a protein similar to nascent polypeptide-associated complex alpha subunit isoform 2, phosphoglycerate kinase 1, glucosidase II, transketolase and alpha-tubulin. These could be sorted into 5 functional groups: glycometabolism, cell proliferation, anti-oxidative stress, inflammatory response and cytoskeleton. Western blot and real-time RT-PCR analysis confirmed the down-regulation of phosphoglycerate kinase 1 (PGK1) and up-regulation of moesin identified by 2D-DIGE. Pathway analysis of these 15 differentially expressed proteins showed that CSFV infection altered the metabolism, cytoskeleton and cell proliferation of PUVECs, and that consequently an inflammatory response was induced.

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P21-activated kinase 1 stimulates colon cancer cell growth and migration/invasion via ERK- and AKT-dependent pathways.

Publication Date: 2010 Sep PMID: 20595063
Authors: Huynh, N. - Liu, K. H. - Baldwin, G. S. - He, H.
Journal: Biochim Biophys Acta

The p21-activated kinase (PAK) family of serine/threonine kinases plays an important role in cell proliferation, survival and motility, as well as in cell transformation and tumor progression. PAK1 promotes transformation through facilitating the ERK/MAPK pathway and enhances cell migration and survival by stimulating AKT. PAK1 expression increases with the progression of colorectal cancer (CRC). In this study, we have investigated the importance of PAK1 in the biology of colon cancer cells. Reduction of PAK1 expression decreased the activities of ERK and AKT leading to decreased cell proliferation, migration/invasion, and survival. Dual inhibition of ERK and AKT suppressed these cellular processes to levels comparable to those achieved by reduction of PAK1 expression, whereas inactivation of either the ERK or AKT pathway alone partially inhibited cell migration/invasion and survival and had no effect on proliferation. We conclude that PAK1 stimulates colon cancer cell proliferation, migration/invasion, and survival via ERK- and AKT-dependent pathways. These findings establish the central importance of PAK1 in CRC signal transduction and clarify the mechanism by which PAK1 regulates CRC growth and migration. Instead of simultaneously inhibiting both ERK and AKT, the PAK1 convergence point could be an alternative target for CRC therapy.

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Phosphatidylcholine as a constituent in the colonic mucosal barrier-Physiological and clinical relevance.

Publication Date: 2010 Sep PMID: 20595010
Authors: Ehehalt, R. - Braun, A. - Karner, M. - Fullekrug, J. - Stremmel, W.
Journal: Biochim Biophys Acta

Phosphatidylcholine (PC) is an important constituent of the gastrointestinal tract. PC molecules are not only important in intestinal cell membranes but also receiving increasing attention as protective agents in the gastrointestinal barrier. They are largely responsible for establishing the hydrophobic surface of the colon. Decreased phospholipids in colonic mucus could be linked to the pathogenesis of ulcerative colitis, a chronic inflammatory bowel disease. Clinical studies revealed that therapeutic addition of PC to the colonic mucus of these patients alleviated the inflammatory activity. This positive role is still elusive, however, we hypothesized that luminal PC has two possible functions: first, it is essential for surface hydrophobicity, and second, it is integrated into the plasma membrane of enterocytes and it modulates the signaling state of the mucosa. The membrane structure and lipid composition of cells is a regulatory component of the inflammatory signaling pathways. In this perspective, we will shortly summarize what is known about the localization and protective properties of PC in the colonic mucosa before turning to its evident medical importance. We will discuss how PC contributes to our understanding of the pathogenesis of ulcerative colitis and how reinforcing the luminal phospholipid monolayer can be used as a therapeutic concept in humans.

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Biochemical characterization of two thymidylate synthases in Corynebacterium glutamicum NCHU 87078.

Publication Date: 2010 Sep PMID: 20595007
Authors: Kan, S. C. - Liu, J. S. - Hu, H. Y. - Chang, C. M. - Lin, W. D. - Wang, W. C. - Hsu, W. H.
Journal: Biochim Biophys Acta

The genome of Corynebacterium glutamicum NCHU 87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His(6)-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414mU mg(-)(1) protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10-day incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3A resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70-73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX.

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Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin-actin interaction.

Publication Date: 2010 Sep PMID: 20595006
Authors: Ferjani, I. - Fattoum, A. - Manai, M. - Benyamin, Y. - Roustan, C. - Maciver, S. K.
Journal: Biochim Biophys Acta

Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478-485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18-28 and 360-372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the alpha-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1.

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Pharmacological profiles of the murine gastric and colonic H,K-ATPases.

Publication Date: 2010 Sep PMID: 20594946
Authors: Shao, J. - Gumz, M. L. - Cain, B. D. - Xia, S. L. - Shull, G. E. - van Driel, I. R. - Wingo, C. S.
Journal: Biochim Biophys Acta

BACKGROUND: The H,K-ATPase, consisting of alpha and beta subunits, belongs to the P-type ATPase family. There are two isoforms of the alpha subunit, HKalpha(1) and HKalpha(2) encoded by different genes. The ouabain-resistant gastric HKalpha(1)-H,K-ATPase is Sch28080-sensitive. However, the colonic HKalpha(2)-H,K-ATPase from different species shows poor primary structure conservation of the HKalpha(2) subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKalpha(1), HKalpha(2), or HKbeta genes. METHODS: Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKalpha(1), HKalpha(2), or HKbeta knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined. RESULTS: The colonic H,K-ATPase demonstrated slightly greater affinity for K(+) than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKalpha(2) KO but was observed in HKbeta KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKalpha(1)-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKalpha(2)-H,K-ATPase was not inhibited by these compounds. CONCLUSIONS: These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride. GENERAL SIGNIFICANCE: Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.

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Identification of an atypical peptidyl-prolyl cis/trans isomerase from trypanosomatids.

Publication Date: 2010 Sep PMID: 20580912
Authors: Erben, E. D. - Valguarnera, E. - Nardelli, S. - Chung, J. - Daum, S. - Potenza, M. - Schenkman, S. - Tellez-Inon, M. T.
Journal: Biochim Biophys Acta

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: k(cat)/K(M)=97.1 /M/s), like that found for human Par14. In contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell.

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Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors.

Publication Date: 2010 Sep PMID: 20580860
Authors: Wang, Z. - Watt, W. - Brooks, N. A. - Harris, M. S. - Urban, J. - Boatman, D. - McMillan, M. - Kahn, M. - Heinrikson, R. L. - Finzel, B. C. - Wittwer, A. J. - Blinn, J. - Kamtekar, S. - Tomasselli, A. G.
Journal: Biochim Biophys Acta

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel beta-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

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Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance.

Publication Date: 2010 Sep PMID: 20580819
Authors: Addo, M. G. - Cossard, R. - Pichard, D. - Obiri-Danso, K. - Rotig, A. - Delahodde, A.
Journal: Biochim Biophys Acta

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown. Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans. This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

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Proteomic analysis of the transitional endoplasmic reticulum in hepatocellular carcinoma: An organelle perspective on cancer.

Publication Date: 2010 Sep PMID: 20576523
Authors: Roy, L. - Laboissiere, S. - Abdou, E. - Thibault, G. - Hamel, N. - Taheri, M. - Boismenu, D. - Lanoix, J. - Kearney, R. E. - Paiement, J.
Journal: Biochim Biophys Acta

The transitional endoplasmic reticulum (tER) is composed of both rough and smooth ER membranes and thus participates in functions attributed to both these two subcellular compartments. In this paper we have compared the protein composition of tER isolated from dissected liver tumor nodules of aflatoxin B1-treated rats with that of tER from control liver. Tandem mass spectrometry (MS), peptide counts and immunoblot validation were used to identify and determine the relative expression level of proteins. Inhibitors of apoptosis (i.e. PGRMC1, tripeptidyl peptidase II), proteins involved in ribosome biogenesis (i.e. nucleophosmin, nucleolin), proteins involved in translation (i.e. eEF-2, and subunits of eIF-3), proteins involved in ubiquitin metabolism (i.e. proteasome subunits, USP10) and proteins involved in membrane traffic (i.e. SEC13-like 1, SEC23B, dynactin 1) were found overexpressed in tumor tER. Transcription factors (i.e. Pur-beta, BTF3) and molecular targets for C-Myc and NF-kappa B were observed overexpressed in tER from tumor nodules. Down-regulated proteins included cytochrome P450 proteins and enzymes involved in fatty acid metabolism and in steroid metabolism. Unexpectedly expression of the protein folding machinery (i.e. calreticulin) and proteins of the MHC class I peptide-loading complex did not change. Proteins of unknown function were detected in association with the tER and the novel proteins showing differential expression are potential new tumor markers. In many cases differential expression of proteins in tumor tER was comparable to that of corresponding genes reported in the Oncomine human database. Thus the molecular profile of tumor tER is different and this may confer survival advantage to tumor cells in cancer.

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Regulatory role of nitric oxide in the reduced survival of erythrocytes in visceral leishmaniasis.

Publication Date: 2010 Sep PMID: 20576500
Authors: Chowdhury, K. D. - Sen, G. - Biswas, T.
Journal: Biochim Biophys Acta

BACKGROUND: Nitric oxide (NO) plays a vital role in maintaining the survivability of circulating erythrocytes. Here we have investigated whether NO depletion associated with visceral leishmaniasis (VL) is responsible for the reduced survival of erythrocytes observed during the disease. METHODS: Infected hamsters were treated with standard anti-leishmanial sodium stibogluconate (SAG) and NO donor isosorbide dinitrate (ISD). Erythrophagocytosis by macrophages was determined by labelling the cells with FITC followed by flow cytometry. Aggregation of band3 was estimated from band3 associated EMA fluorescence. Caspase 3 activity was measured using immunosorbent assay kit. Phosphatidylserine (PS) externalization and cell shrinkage were determined using annexin V. Aminophspholipid translocase and scramblase activities were measured following NBD-PS and NBD-PC internalization, respectively. RESULTS: Impairment of both synthesis and uptake of NO resulted in decreased bioavailability of this signaling molecule in erythrocytes in VL. NO level was replenished after simultaneous treatment with ISD and SAG. Combination treatment decreased red cell apoptosis in infected animals by deactivating caspase 3 through s-nitrosylation. Drug treatment prevented infection-mediated ATP depletion and altered calcium homeostasis in erythrocytes. Improved metabolic environment effectively amended dysregulation of aminophospholipid translocase and scramblase, which in turn reduced cell shrinkage, and exposure of phosphatidylserine on the cell surface under the diseased condition. CONCLUSION AND GENERAL SIGNIFICANCE: In this study, we have identified NO depletion to be an important factor in promoting premature hemolysis with the progress of leishmanial infection. The study implicates NO to be a possible target for future drug development towards the promotion of erythrocyte survival in VL.

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The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition.

Publication Date: 2010 Sep PMID: 20570648
Authors: Khemtemourian, L. - Engel, M. F. - Liskamp, R. M. - Hoppener, J. W. - Killian, J. A.
Journal: Biochim Biophys Acta

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet beta-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP(1-19), plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP(1)(-)(19) in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP(1-19) is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP(1-19) is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.

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Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity.

Publication Date: 2010 Sep PMID: 20570635
Authors: Aye, I. L. - Waddell, B. J. - Mark, P. J. - Keelan, J. A.
Journal: Biochim Biophys Acta

ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 mediate the efflux of cholesterol and other sterols. Both transporters are expressed on the fetal capillaries of the placenta and are involved in maternal-to-fetal cholesterol delivery. In this study, we report that ABCA1 and ABCG1 are also present on the syncytiotrophoblast, the maternal facing placental membrane. Syncytial ABCA1 expression is apical, suggesting a role in cholesterol efflux to the mother, while ABCG1 is expressed basolaterally indicating transport to the fetus. Silencing of ABCA1 expression in primary trophoblasts in culture, or pharmacological antagonism by glyburide, decreased cholesterol efflux to apolipoprotein A-I (apoA-I) compared to controls, while ABCG1-silencing decreased cholesterol efflux to high density lipoproteins (HDL). In contrast, treatment with endogenous or synthetic LXR alpha/beta ligands such as T0901317 increased ABCA1 and ABCG1 expression and enhanced cholesterol efflux to apoA-I and HDL, respectively, while treatment with pharmacological PPAR-alpha or -gamma ligands was without effect. Trophoblasts transfected with ABCA1 or ABCG1 siRNA were more sensitive to toxic oxysterols substrates (25-hydroxycholesterol and 7-ketocholesterol) compared to mock-transfected cells, while prior treatment with T0901317 reduced oxysterol-mediated toxicity. These results identify syncytial ABCA1 and ABCG1 as important, inducible cholesterol transporters which also prevent placental accumulation of cytotoxic oxysterols.

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Signal transduction pathways associated with ATP-induced proliferation of colon adenocarcinoma cells.

Publication Date: 2010 Sep PMID: 20562007
Authors: Buzzi, N. - Boland, R. - Russo de Boland, A.
Journal: Biochim Biophys Acta

BACKGROUND: In previous work, we have demonstrated that extracellular adenosine 5'-triphosphate (ATP) acts on intestinal Caco-2 cell P2Y receptors promoting a rapid increase in the phosphorylation of ERK1/2, p46 JNK and p38 MAP kinases (MAPKs). METHODS AND RESULTS: In this study, we investigated whether the extracellular ATP-P2Y receptor signalling pathways were required for the proliferation of Caco-2 cells. Confocal microscopy and immunobloting studies showed that ERK1/2 and JNK translocate into the nucleus of the cells stimulated by ATP, where they participate, together with p38 MAPK, in the phosphorylation of JunD, ATF-1 and ATF-2 transcription factors. In addition, ATP through the activation of MAPKs induces the expression of the immediate early genes products of the Jun family, c-Fos and MAP kinase phosphatase-1 (MKP-1). Moreover, ERK1/2 and p38 MAPK are involved in the phosphorylation of MKP-1 in Caco-2 cells. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, ATP increased Caco-2 cell proliferation, and this effect was blocked by UO126, SB203580 and SP600125, the specific inhibitors of ERK1/2, p38 MAPK and JNK1/2, respectively. CONCLUSION: Extracellular ATP induces proliferation of Caco-2 human colonic cancer cells by activating MAPK cascades and modulation of transcription factors. GENERAL SIGNIFICANCE: These findings and identification of the specific P2Y subtype receptors involved in the mitogenic effect of ATP on Caco-2 cells might be relevant for understanding tumor cell development, resistance to treatment regimens and the design of new therapeutic strategies.

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High-affinity binding of seminal plasma PSP94 to human immunoglobulin is through the Fab domain.

Publication Date: 2010 Sep PMID: 20554063
Authors: Jagtap, D. D. - Modi, D. N. - Kumar, M. - Pathak, B. R. - Mahale, S. D.
Journal: Biochim Biophys Acta

Prostate secretory protein of 94 amino acids (PSP94) is one of the major proteins present in human seminal plasma. We had earlier reported that PSP94 has the ability to bind to human IgG. The aims of the present study were to further delineate the PSP94-IgG interaction and to understand whether this could have any significance in sperm function. Direct binding of IgG fragments to PSP94 showed maximal binding with F(ab')(2) followed by Fab, while Fc displayed least binding in ELISA. Binding kinetics of PSP94-IgG interaction using surface plasmon resonance (SPR) revealed high-affinity binding of IgG to PSP94 with a dissociation constant (K(D)) of 8.8x10(-)(11)M. PSP94-IgG interaction was found to be through the Fab domains of IgG. Real-time interaction kinetics revealed association constants for binding of IgG, Fab, and F(ab')(2) towards PSP94 to be of the same order but with altered dissociation constants. IgG and its F(ab')(2) fragment once complexed to PSP94 demonstrated negligible dissociation, while dissociation rate of Fab fragment was 6.6x10(-)(4). In silico molecular modeling of PSP94-IgG complex identified N- and C-terminal beta-strands of PSP94 to be the most plausible region involved in IgG interaction. Immunofluorescence studies revealed that IgG bound to human spermatozoa predominantly in the tail region, which could be prevented when IgG was preincubated with PSP94. This study reports for the first time that IgG forms a high-affinity complex with PSP94 through its F(ab')(2) domain and reveals the ability of PSP94 to prevent binding of IgG to spermatozoa.

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Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomycescerevisiae.

Publication Date: 2010 Sep PMID: 20554061
Authors: Heier, C. - Taschler, U. - Rengachari, S. - Oberer, M. - Wolinski, H. - Natter, K. - Kohlwein, S. D. - Leber, R. - Zimmermann, R.
Journal: Biochim Biophys Acta

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Delta mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.

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Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of tripeptidyl peptidase II.

Publication Date: 2010 Sep PMID: 20553980
Authors: Villasevil, E. M. - Guil, S. - Lopez-Ferreras, L. - Sanchez, C. - Del Val, M. - Anton, L. C.
Journal: Biochim Biophys Acta

In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.

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Effects of CYP7B1-mediated catalysis on estrogen receptor activation.

Publication Date: 2010 Sep PMID: 20553962
Authors: Pettersson, H. - Lundqvist, J. - Norlin, M.
Journal: Biochim Biophys Acta

Most of the many biological effects of estrogens are mediated via the estrogen receptors ERalpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERbeta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3beta,17beta-diol (Aene-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7alpha-hydroxy-DHEA, previously suggested to affect ERbeta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3beta-Adiol. CYP7B1-mediated metabolism of 3beta-Adiol has been proposed to influence ERbeta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERalpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7alpha-hydroxymetabolite will result in loss of action.

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Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31-43 but not of peptide 57-68 by epithelial cells.

Publication Date: 2010 Sep PMID: 20553859
Authors: Caputo, I. - Barone, M. V. - Lepretti, M. - Martucciello, S. - Nista, I. - Troncone, R. - Auricchio, S. - Sblattero, D. - Esposito, C.
Journal: Biochim Biophys Acta

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31-43 (but not peptide 57-68) uptake by cells, thereby impairing the ability of p31-43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31-43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.

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Activation of ASK1, downstream MAPKK and MAPK isoforms during cardiac ischaemia.

Publication Date: 2010 Sep PMID: 20550965
Authors: Harding, S. J. - Browne, G. J. - Miller, B. W. - Prigent, S. A. - Dickens, M.
Journal: Biochim Biophys Acta

p38 MAPK is activated potently during cardiac ischaemia, although the precise mechanism by which it is activated is unclear. We used the isolated perfused rat heart to investigate the signalling pathways activated upstream of p38 during global cardiac ischaemia. Ischaemia strongly activated p38alpha but not the JNK pathway. The MAPKKs, MKK3, MKK4 and MKK6 have previously been identified as potential upstream activators of p38; however, in the ischaemic perfused heart, we saw activation of MKK3 and MKK6 but not MKK4. MKK3 and MKK6 showed different temporal patterns of activity, indicating distinct modes of activation and physiological function. Consistent with a lack of JNK activation, we saw no activation of MKK4 or MKK7 at any time point during ischaemia. A lack of MKK4 activation indicates, at least in the ischaemic heart, that MKK4 is not a physiologically relevant activator of p38. The MAPKKK, ASK1, was strongly activated late during ischaemia, with a similar time course to that of MKK6 and in ischaemic neonatal cardiac myocytes ASK1 expression preferentially activated MKK6 rather than MKK3. These observations suggest that during ischaemia ASK1 is coupled to p38 activation primarily via MKK6. Potent activation of ASK1 during ischaemia without JNK activation shows that during cardiac ischaemia, ASK1 preferentially activates the p38 pathway. These results demonstrate a specificity of responses seldom seen in previous studies and illustrate the benefits of using direct assays in intact tissues responding to physiologically relevant stimuli to unravel the complexities of MAPK signalling.

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Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na(+)/Ca(2+) exchange.

Publication Date: 2010 Sep PMID: 20550942
Authors: Zhdanov, A. V. - Ward, M. W. - Taylor, C. T. - Souslova, E. A. - Chudakov, D. M. - Prehn, J. H. - Papkovsky, D. B.
Journal: Biochim Biophys Acta

Fluctuating extracellular Ca(2+) regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca(2+) from 1.8 to
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Analysis of TSC1 truncations defines regions involved in TSC1 stability, aggregation and interaction.

Publication Date: 2010 Sep PMID: 20547222
Authors: Hoogeveen-Westerveld, M. - Exalto, C. - Maat-Kievit, A. - van den Ouweland, A. - Halley, D. - Nellist, M.
Journal: Biochim Biophys Acta

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the target of rapamycin complex 1 (TORC1). Here we investigate TSC1 structure and function by analysing a series of truncated TSC1 proteins. We identify specific regions of the protein that are important for TSC1 stability, localisation, interactions and function.

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Glutaredoxin 2 prevents H(2)O(2)-induced cell apoptosis by protecting complex I activity in the mitochondria.

Publication Date: 2010 Oct PMID: 20547138
Authors: Wu, H. - Xing, K. - Lou, M. F.
Journal: Biochim Biophys Acta

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I.

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Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study.

Publication Date: 2010 Sep PMID: 20547137
Authors: Birungi, M. - Folea, M. - Battchikova, N. - Xu, M. - Mi, H. - Ogawa, T. - Aro, E. M. - Boekema, E. J.
Journal: Biochim Biophys Acta

Cyanobacterial NDH-1 is a multisubunit complex involved in proton translocation, cyclic electron flow around photosystem I and CO(2) uptake. The function and location of several of its small subunits are unknown. In this work, the location of the small subunits NdhL, -M, -N, -O and CupS of Synechocystis 6803 NDH-1 was established by electron microscopy (EM) and single particle analysis. To perform this, the subunits were enlarged by fusion with the YFP protein. After classification of projections, the position of the YFP tag was revealed; all five subunits are integrated in the membrane domain. The results on NDH-1 demonstrate that a GFP tag can be revealed after data processing of EM data sets of moderate size, thus showing that this way of labeling is a fast and reliable way for subunit mapping in multisubunit complexes after partial purification.

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Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable.

Publication Date: 2010 Sep PMID: 20546701
Authors: Kumar, A. - Shukla, S. - Mandal, A. - Shukla, S. - Ambudkar, S. V. - Prasad, R.
Journal: Biochim Biophys Acta

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.

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Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

Publication Date: 2010 Sep PMID: 20542090
Authors: Ito, K. - Sugawara, T. - Koizumi, A. - Nakajima, K. I. - Shimizu-Ibuka, A. - Shiroishi, M. - Asada, H. - Yurugi-Kobayashi, T. - Shimamura, T. - Asakura, T. - Masuda, K. - Ishiguro, M. - Misaka, T. - Iwata, S. - Kobayashi, T. - Abe, K.
Journal: Biochim Biophys Acta

BACKGROUND: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS: As a result, a 2mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.

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Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems.

Publication Date: 2010 Sep PMID: 20542013
Authors: Madsen, H. B. - Arboe-Andersen, H. M. - Rozlosnik, N. - Madsen, F. - Ifversen, P. - Kasimova, M. R. - Nielsen, H. M.
Journal: Biochim Biophys Acta

The modified ISCOMs, so-called Posintro nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Forster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro nanoparticles leads to enhanced disruption of lipid organization.

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Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study.

Publication Date: 2010 Sep PMID: 20542012
Authors: Ponomarenko, N. S. - Poluektov, O. G. - Bylina, E. J. - Norris, J. R.
Journal: Biochim Biophys Acta

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D(+) couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.

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Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

Publication Date: 2010 Sep PMID: 20541621
Authors: De Geronimo, E. - Hagan, R. M. - Wilton, D. C. - Corsico, B.
Journal: Biochim Biophys Acta

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.

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Regulation of post-translational protein arginine methylation during HeLa cell cycle.

Publication Date: 2010 Sep PMID: 20541591
Authors: Kim, C. - Lim, Y. - Yoo, B. C. - Won, N. H. - Kim, S. - Kim, G.
Journal: Biochim Biophys Acta

BACKGROUND: Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle. METHODS: The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies. RESULTS: Proteins with approximate molecular masses of 80kDa, 68kDa, and 64kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80kDa and aDMA-68kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64kDa proteins as cleavage stimulation factor 64kDa subunit (CstF-64), and sDMA-25kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI. CONCLUSION: Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs. GENERAL SIGNIFICANCE: These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle.

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Erythrocyte morphological states, phases, transitions and trajectories.

Publication Date: 2010 Sep PMID: 20538541
Authors: Rudenko, S. V.
Journal: Biochim Biophys Acta

Morphological response (MR) of red blood cells represents a triphasic sequence of spontaneously occurring shape transformation between different shape states upon transfer the cells into isotonic sucrose solution in the order: S(0) (initial discoid shape in physiological saline)-->S(1) (echinocytic shape at the beginning of MR, phase 1)-->S(2) (intermediate discoid shape, phase 2)-->S(3) (final stomatocytic shape, phase 3). In this paper, the dynamics of cell shape changes was investigated by non-invasive light fluctuation method and optical microscopy. Among 12 possible transitions between four main shape states, we experimentally demonstrate here an existence of nine transitions between neighbour or remote states in this sequence. Based on these findings and data from the literature, we may conclude that red blood cells are able to change their shape through direct transitions between four main states except transition S(1)-->S(0), which has not been identified yet. Some shape transitions and their temporal sequence are in accord with predictions of bilayer couple concept, whereas others for example transitions between remote states S(3)-->S(1), S(1)-->S(3) and S(3)-->S(0) are difficult to explain based solely on the difference in relative surface areas of both leaflets of membrane suggesting more complex mechanisms involved. Our data show that MR could represents a phenomenon in which the major role can play pH and chloride-sensitive sensor and switching mechanisms coupled with transmembrane signaling thus involving both cytoskeleton and membrane in coordinated shape response on changes in cell ionic environment.

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Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom.

Publication Date: 2010 Sep PMID: 20538077
Authors: Peichoto, M. E. - Leme, A. F. - Pauletti, B. A. - Batista, I. C. - Mackessy, S. P. - Acosta, O. - Santoro, M. L.
Journal: Biochim Biophys Acta

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 degrees C, primarily producing a approximately 32.6kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca(2+), patagonfibrase was only partially autolysed, giving rise mainly to one fragment of approximately 52.2kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.

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Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1).

Publication Date: 2010 Sep PMID: 20538076
Authors: Varejao, N. - Almeida, M. D. - De Cicco, N. N. - Atella, G. C. - Coelho, L. C. - Correia, M. A. - Foguel, D.
Journal: Biochim Biophys Acta

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.

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Getting the mOST from OST: Role of organic solute transporter, OSTalpha-OSTbeta, in bile acid and steroid metabolism.

Publication Date: 2010 Sep PMID: 20538072
Authors: Dawson, P. A. - Hubbert, M. L. - Rao, A.
Journal: Biochim Biophys Acta

The organic solute transporter (OST)(alpha)-OST(beta) is an unusual heteromeric carrier expressed in a variety of tissues including the small intestine, colon, liver, biliary tract, kidney, and adrenal gland. In polarized epithelial cells, OSTalpha-OSTbeta protein is localized on the basolateral membrane and functions in the export or uptake of bile acids and steroids. This article reviews recent results including studies of knockout mouse models that provide new insights to the role of OSTalpha-OSTbeta in the compartmentalization and metabolism of these important lipids.

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Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling.

Publication Date: 2010 Sep PMID: 20538056
Authors: Violante, S. - Ijlst, L. - van Lenthe, H. - de Almeida, I. T. - Wanders, R. J. - Ventura, F. V.
Journal: Biochim Biophys Acta

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K(i) of 18.8muM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.

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Decline in mitochondrial bioenergetics and shift to ketogenic profile in brain during reproductive senescence.

Publication Date: 2010 Oct PMID: 20538040
Authors: Yao, J. - Hamilton, R. T. - Cadenas, E. - Brinton, R. D.
Journal: Biochim Biophys Acta

BACKGROUND: We have previously demonstrated that mitochondrial bioenergetic deficits precede Alzheimer's pathology in the female triple transgenic Alzheimer's (3xTgAD) mouse model. Herein, we sought to determine the impact of reproductive senescence on mitochondrial function in the normal non-transgenic (nonTg) and 3xTgAD female mouse model of AD. METHODS: Both nonTg and 3xTgAD female mice at 3, 6, 9, and 12months of age were sacrificed and mitochondrial bioenergetic profile as well as oxidative stress markers were analyzed. RESULTS: In both nonTg and 3xTgAD mice, reproductive senescence paralleled a significant decline in PDH, and Complex IV cytochrome c oxidase activity and mitochondrial respiration. During the reproductive senescence transition, both nonTg and 3xTgAD mice exhibited greater individual variability in bioenergetic parameters suggestive of divergent bioenergetic phenotypes. Following transition through reproductive senescence, enzymes required for long-chain fatty acid (HADHA) and ketone body (SCOT) metabolism were significantly increased and variability in cytochrome c oxidase (Complex IV) collapsed to cluster at a approximately 40% decline in both the nonTg and 3xTgAD brain which was indicative of alternative fuel generation with concomitant decline in ATP generation. CONCLUSIONS: These data indicate that reproductive senescence in the normal nonTg female brain parallels the shift to ketogenic/fatty acid substrate phenotype with concomitant decline in mitochondrial function and exacerbation of bioenergetic deficits in the 3xTgAD brain. GENERAL SIGNIFICANCE: These findings provide a plausible mechanism for increased life-time risk of AD in postmenopausal women and suggest an optimal window of opportunity to prevent or delay decline in bioenergetics during reproductive senescence.

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Quantitation of cholesterol incorporation into extruded lipid bilayers.

Publication Date: 2010 Sep PMID: 20537979
Authors: Ibarguren, M. - Alonso, A. - Tenchov, B. G. - Goni, F. M.
Journal: Biochim Biophys Acta

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.

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Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy.

Publication Date: 2010 Sep PMID: 20529691
Authors: Rappaport, F. - Zhang, J. - Vos, M. H. - Gennis, R. B. - Borisov, V. B.
Journal: Biochim Biophys Acta

Cytochrome bd is a terminal quinol:O(2) oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b(558), b(595), and d. The role of heme b(595) remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b(595) causing loss of an absorption band centered at 435nm, thus supporting interactions between heme b(595) and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with tau approximately 12mus, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with tau approximately 14ns, 14mus, and 280mus. The spectra of the absorption changes associated with these components are different in line shape. The 14ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-mus component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in approximately 4% of the enzyme population. The final, 280-mus component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b(595), and not that of heme b(558), controls the pathway(s) by which CO migrates between heme d and the medium.

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P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells.

Publication Date: 2010 Sep PMID: 20529664
Authors: Constantinescu, P. - Wang, B. - Kovacevic, K. - Jalilian, I. - Bosman, G. J. - Wiley, J. S. - Sluyter, R.
Journal: Biochim Biophys Acta

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium(+) uptake into MEL cells in a concentration-dependent fashion and with an EC(50) of approximately 154muM. The most potent P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium(+) uptake. ATP-induced ethidium(+) and YO-PRO-1(2+) uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.

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Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels.

Publication Date: 2010 Sep PMID: 20529663
Authors: Caldwell, D. B. - Malcolm, H. R. - Elmore, D. E. - Maurer, J. A.
Journal: Biochim Biophys Acta

Studies of bacterial ion channels have provided significant insights into the structure-function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.

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Adhesive and conformational behaviour of mycolic acid monolayers.

Publication Date: 2010 Sep PMID: 20529662
Authors: Zhang, Z. - Pen, Y. - Edyvean, R. G. - Banwart, S. A. - Dalgliesh, R. M. - Geoghegan, M.
Journal: Biochim Biophys Acta

We have studied the pH-dependent interaction between mycolic acid (MA) monolayers and hydrophobic and hydrophilic surfaces using molecular (colloidal probe) force spectroscopy. In both cases, hydrophobic and hydrophilic monolayers (prepared by Langmuir-Blodgett and Langmuir-Schaefer deposition on silicon or hydrophobized silicon substrates, respectively) were studied. The force spectroscopy data, fitted with classical DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory to examine the contribution of electrostatic and van der Waals forces, revealed that electrostatic forces are the dominant contribution to the repulsive force between the approaching colloidal probe and MA monolayers. The good agreement between data and the DLVO model suggest that beyond a few nm away from the surface, hydrophobic, hydration, and specific chemical bonding are unlikely to contribute to any significant extent to the interaction energy between the probe and the surface. The pH-dependent conformation of MA molecules in the monolayer at the solid-liquid interface was studied by ellipsometry, neutron reflectometry, and with a quartz crystal microbalance. Monolayers prepared by the Langmuir-Blodgett method demonstrated a distinct pH-responsive behaviour, while monolayers prepared by the Langmuir-Schaefer method were less sensitive to pH variation. It was found that the attachment of water molecules plays a vital role in determining the conformation of the MA monolayers.

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Determination of the intrinsic redox potentials of FeS centers of respiratory complex I from experimental titration curves.

Publication Date: 2010 Sep PMID: 20513348
Authors: Medvedev, E. S. - Couch, V. A. - Stuchebrukhov, A. A.
Journal: Biochim Biophys Acta

Recently, Euro et al. [Biochem. 47, 3185 (2008) ] have reported titration data for seven of nine FeS redox centers of complex I from Escherichiacoli. There is a significant uncertainty in the assignment of the titration data. Four of the titration curves were assigned to N1a, N1b, N6b, and N2 centers; one curve either to N3 or N7; one more either to N4 or N5; and the last one denoted Nx could not be assigned at all. In addition, the assignment of the titration data to the N6b/N6a pair is also uncertain. In this paper, using our calculated interaction energies [Couch et al. BBA 1787, 1266 (2009)], we perform statistical analysis of these data, considering a variety of possible assignments, find the best fit, and determine the intrinsic redox potentials of the centers. The intrinsic potentials could be determined with an uncertainty of less than +/-10mV at a 95% confidence level for best fit assignments. We also find that the best agreement between theoretical and experimental titration curves is obtained with the N6b-N2 interaction equal to 71+/-14 or 96+/-26mV depending on the N6b/N6a titration data assignment, which is stronger than was expected and may indicate a close distance of the N2 center to the membrane surface.

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Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states.

Publication Date: 2010 Sep PMID: 20510669
Authors: Barucha-Kraszewska, J. - Kraszewski, S. - Jurkiewicz, P. - Ramseyer, C. - Hof, M.
Journal: Biochim Biophys Acta

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.

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Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport.

Publication Date: 2010 Sep PMID: 20510358
Authors: Shi, P. - Strom, A. L. - Gal, J. - Zhu, H.
Journal: Biochim Biophys Acta

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein-dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.

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Mouse models of neurological disorders-A comparison of heritable and acquired traits.

Publication Date: 2010 Oct PMID: 20510357
Authors: Harper, A.
Journal: Biochim Biophys Acta

Human neurological disorders include a wide range of illnesses which have a disproportionately high prevalence in the increasingly populous geriatric community. Any research effort directed at discovering the aetiology of neurological disease is greatly enhanced with in vivo models of the disease of interest. Scientific research incorporating the use of mice has advanced rapidly in the last three decades. Relatively simple to breed, maintain and train, mice have many advantages over other species for use in research. More than a century of selective breeding has provided investigators with a rich gene pool and sub-strain diversity from which to choose for their research. Thus the dramatic increase in genetic screening and gene engineering that has occurred in research in recent decades has enabled the generation of a multitude of mouse models. This review discusses the relative utility of mouse models in which a heritable or non-heritable (acquired) manipulation has been used to model a specified trait of a human neurological disorder. The techniques used in deriving useful genetic alterations or modifications and in generating acquired mouse models are outlined with examples of each provided.

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Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes.

Publication Date: 2010 Sep PMID: 20510172
Authors: Rokitskaya, T. I. - Sumbatyan, N. V. - Tashlitsky, V. N. - Korshunova, G. A. - Antonenko, Y. N. - Skulachev, V. P.
Journal: Biochim Biophys Acta

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain (n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10-12 and substantially decreased at n=16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n=16 or n=18 as compared to that with n=12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n=18 and n=12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.

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Potential role of the membrane in hERG channel functioning and drug-induced long QT syndrome.

Publication Date: 2010 Sep PMID: 20510171
Authors: Chartrand, E. - Arnold, A. A. - Gravel, A. - Jenna, S. - Marcotte, I.
Journal: Biochim Biophys Acta

The human ether-a-go-go related gene (hERG) potassium channels are located in the myocardium cell membrane where they ensure normal cardiac activity. The binding of drugs to this channel, a side effect known as drug-induced (acquired) long QT syndrome (ALQTS), can lead to arrhythmia or sudden cardiac death. The hERG channel is a unique member of the family of voltage-gated K(+) channels because of the long extracellular loop connecting its transmembrane S5 helix to the pore helix in the pore domain. Considering the proximal position of the S5-P linker to the membrane surface, we have investigated the interaction of its central segment I(583)-Y(597) with bicelles. Liquid and solid-state NMR experiments as well as circular dichroism results show a strong affinity of the I(583)-Y(597) segment for the membrane where it would sit on the surface with no defined secondary structure. A structural dependence of this segment on model membrane composition was observed. A helical conformation is favoured in detergent micelles and in the presence of negative charges. Our results suggest that the interaction of the S5-P linker with the membrane could participate in the stabilization of transient channel conformations, but helix formation would be triggered by interactions with other hERG domains. Because potential drug binding sites on the S5-P linker have been identified, we have explored the role of this segment in ALQTS. Four LQTS-liable drugs were studied which showed more affinity for the membrane than this hERG segment. Our results, therefore, identify two possible roles for the membrane in channel functioning and ALQTS.

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Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state.

Publication Date: 2010 Sep PMID: 20510170
Authors: Thalhammer, A. - Hundertmark, M. - Popova, A. V. - Seckler, R. - Hincha, D. K.
Journal: Biochim Biophys Acta

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly alpha-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic alpha-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.

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Expression, purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2.

Publication Date: 2010 Sep PMID: 20493972
Authors: Lu, B. - Zhai, Y. - Wu, C. - Pang, X. - Xu, Z. - Sun, F.
Journal: Biochim Biophys Acta

Leucine-rich repeat kinase 2 gene is a key factor for Parkinson's disease and encodes for a large protein kinase LRRK2 (280kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2's N-terminal fragments N1 (aa12-320) and N2 (aa12-860). The purified N2 protein was identified by mass spectrometry and N1's molecular weight was determined to be 33.23kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1's multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both alpha helixes and beta sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.

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Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: Implication of PARP-1, APEX-1 and ERK1/2 catalytic activities.

Publication Date: 2010 Sep PMID: 20493910
Authors: Levros, L. C. Jr - Carmo, S. D. - Edouard, E. - Legault, P. - Charfi, C. - Rassart, E.
Journal: Biochim Biophys Acta

Human Apolipoprotein D (apoD) is upregulated under several stress conditions and pathological situations such as neurodegenerative diseases and cancers. We previously showed that apoD mRNA expression is induced in growth-arrested cells and demonstrated the specific binding of nuclear proteins to the region -514 to -475 of the promoter. Such region contains a pair of Serum Responsive Elements (SRE), an Ets-Binding Site (EBS) and a Glucocorticoid Responsive Element (GRE). In this study, we show that Parp-1, HnRNP-U, CBF-A, BUB-3, Kif4, APEX-1 and Ifi204 bind these regulatory elements of the apoD promoter. Specific binding of HnRNP-U and Parp-1 was confirmed by Electrophoretic Mobility Shift Assay (EMSA). In a biotin pull-down assay, Kif4 and BUB-3 bind preferentially the SRE1 and the EBS-GRE sites, respectively, while APEX-1 seems recruited indirectly to these elements. We found that the mRNA expression of some of these binding factors is upregulated in growth-arrested cells and that these proteins also transactivate the apoD promoter. In agreement with these results, mutants of APEX-1 and of Parp-1 defective for their DNA-binding and catalytic activities could not transactivate the promoter. The knockdown of Parp-1 and HnRNP-U and the use of specific inhibitors of MEK1/2 and of Parp-1 also inhibited the induction of apoD gene expression. Moreover, ERK1/2 was found activated in a biphasic manner post serum-starvation and the inhibition of Parp-1 causes a sustained activation of ERK2 but not ERK1 for up to 2h. Altogether, these findings demonstrate the importance of Parp-1, APEX-1 and ERK1/2 catalytic activities in the growth arrest-induced apoD gene expression.

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Morgana/CHP-1 is a novel chaperone able to protect cells from stress.

Publication Date: 2010 Sep PMID: 20493909
Authors: Michowski, W. - Ferretti, R. - Wisniewska, M. B. - Ambrozkiewicz, M. - Beresewicz, M. - Fusella, F. - Skibinska-Kijek, A. - Zablocka, B. - Brancaccio, M. - Tarone, G. - Kuznicki, J.
Journal: Biochim Biophys Acta

Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.

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Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles.

Publication Date: 2010 Sep PMID: 20493165
Authors: Kamiya, K. - Kobayashi, J. - Yoshimura, T. - Tsumoto, K.
Journal: Biochim Biophys Acta

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic beta(2) receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.

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