1.
DHHC PROTEIN S-ACYLTRANSFERASES USE A SIMILAR PING-PONG KINETIC MECHANISM BUT DISPLAY DIFFERENT ACYL-COA SPECIFICITIES.
Source
Cornell University, United States.
Abstract
DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHCproteins. A high performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation (PAT) activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of Vmax and Km values for both the peptide N-myristoylated-GCG and palmitoyl-Coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl-enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl-chains 14-carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl-chain transfer to protein substrate, were reduced with stearoyl-CoA compared to palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteinsand may account for the differential S-acylation of proteins observed in cells.
- PMID:
- 22247542
- [PubMed - as supplied by publisher]
Candidate Serum Biomarkers for Prostate Adenocarcinoma Identified by mRNA Differences in Prostate Tissue and Verified with Protein Measurements in Tissue and Blood.
Source
Department of Health Sciences Research.
Abstract
BACKGROUND:
Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation.
METHODS:
We identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue and confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. We analyzed tissue extracts with tandem mass spectrometry (MS/MS) and measured blood concentrations using immunoassays and MS/MS of trypsin-digested, immunoextracted peptides.
RESULTS:
We selected 35 novel candidate prostate adenocarcinoma biomarkers. For all 13 markers having commercial antisera for IHC, tissue expression was confirmed; 6 showed statistical discrimination between nondiseased and malignant tissue, and only 5 were detected in tissue extracts by MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of 8 biomarkers measured by ELISA and 3 of 10 measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared with controls.
CONCLUSION:
Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased concentrations in blood from men with advanced prostate adenocarcinoma compared with controls: apolipoprotein C1 (APOC1), asporin (ASPN), cartilage oligomeric matrix protein (COMP), chemokine (C-X-C motif) ligand 11 (CXCL11), CXCL9, coagulation factor V (F5), and proprotein convertase subtilisin/kexin 6 (PCSK6).
- PMID:
- 22247499
- [PubMed - as supplied by publisher]
Interaction of MxiG with the cytosolic complex of the type III secretion system controls Shigella virulence.
Source
*Department of Cellular Microbiology and.
Abstract
Gram-negative bacteria use the type 3 secretion system (T3SS) to colonize host cells. T3SSs are ring-shaped macromolecular complexes specific for the transport of effector molecules into host cells. It was recently suggested that a cytosolic ring-shaped protein complex delivers effector molecules to the T3SS. However, how transport of effectorproteins is regulated is not known. Here, we report the high-resolution X-ray crystal structure of the whole cytosolic domain of MxiG (MxiG(1-126)), a major component of the inner T3SS rings in Shigella flexneri. MxiG(1-126) folds as an FHA domain, which specifically binds phosphorylated threonines. Indeed, MxiG(1-126) binds to Spa33, a cytoplasmic-ring component of Shigella, as revealed in pulldown studies. Surface plasmon resonance analysis showed specific interaction of MxiG with a Spa33 peptide only if phosphorylated. In total, 24 copies of the MxiG(1-126) crystal structure were fitted into the cryo-EM map of the Shigella T3SS. The phosphoprotein binding site of each MxiG molecule faces the channel of the T3SS, allowing interaction with cytosolic binding partners. Secretion assays and host cell invasion studies of complemented Shigella knockout cells indicated that the phosphoprotein binding of MxiG is essential for bacterial virulence. Our findings suggest that MxiG is involved in T3SS regulation.-Barison, N., Lambers, J., Hurwitz, R., Kolbe, M. Interaction of MxiG with the cytosolic complex of the type III secretion system controls Shigella virulence.
- PMID:
- 22247334
- [PubMed - as supplied by publisher]
CD36 Ectodomain Phosphorylation Blocks Thrombospondin-1 Binding: Structure-Function Relationships and Regulation by Protein Kinase C.
Source
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland OH.
Abstract
OBJECTIVE:
CD36 phosphorylation on its extracellular domain inhibits binding of thrombospondin-1. The mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells are not known. We determined structure-function relationships of CD36 phosphorylation related to thrombospondin-1 peptide binding in vitro and explored mechanisms regulating phosphorylation by protein kinase C (PKC) in melanoma cells.
METHODS AND RESULTS:
Phosphorylation of CD36 peptide on Thr92 by PKCα suppressed binding of thrombospondin-1 peptides in vitro, and the level of inhibition correlated with the level of phosphorylation. Basal phosphorylation levels of CD36 in vivo in platelets, endothelial cells, and melanoma cells were assessed by immunoprecipitation and immunoblot and were found to be very low. Treatment of CD36-transfected melanoma cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, induced substantial CD36 phosphorylation and decreased ligand-mediated recruitment of Src-family proteins to CD36. PMA treatment did not induce detectable extracellular or cell surface-associated kinase activity, and both cycloheximide and brefeldin A blocked CD36 phosphorylation.
CONCLUSIONS:
New protein synthesis and trafficking through the Golgi are required for PMA-induced CD36 phosphorylation, suggesting that phosphorylation probably occurs intracellularly. These studies suggest a novel in vivo pathway for CD36 phosphorylation that modulates cellular affinity for thrombospondin-related proteins to blunt vascular cell signaling.
- PMID:
- 22247259
- [PubMed - as supplied by publisher]
SulE, a Sulfonylurea Herbicide De-esterification Esterase from Hansschlegelia zhihuaiae S113.
Source
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
Abstract
De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of the sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1194 bp, and a putative signalpeptide at the N-terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361 residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides, which gave rise to the corresponding herbicidally inactive parent acids and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in sulfonylurea herbicides' resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicides' metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.
- PMID:
- 22247165
- [PubMed - as supplied by publisher]
Electron Transfer Dissociation Mass Spectrometry in Proteomics.
Source
McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Abstract
Mass spectrometry has rapidly evolved to become the platform of choice for proteomic analysis. While CID remains the major fragmentation method for peptide sequencing, electron transfer dissociation (ETD) is emerging as a complementary method for characterization of peptides and post-translational modifications (PTMs). Here, we review the evolution of ETD and some of its newer applications including characterization of PTMs, non-tryptic peptides and intactproteins. We will also discuss some of the unique features of ETD such as its complementarity with CID and the use of alternating CID/ETD along with issues pertaining to analysis of ETD data. The potential of ETD for applications such as multiple reaction monitoring and proteogenomics in the future will also be discussed.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22246976
- [PubMed - as supplied by publisher]
Combinatorial Peptide Ligand Libraries For The Analysis Of Low-ExpressionProteins. Validation For Normal Urine And Definition Of A First Protein Map.
Source
Laboratory of Pathophysiology of Uremia, G. Gaslini Children Hospithal, Genoa, Italy.
Abstract
In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorialpeptide ligand libraries (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are: 1- definition of the urine sample requirements, 2- optimization of the urine/ligand ratio, 3- essay conditions, 4- en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter-and intra-essay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC- MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly-developed studies on human diseases.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22246922
- [PubMed - as supplied by publisher]
Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay.
Source
Biomarker Department, BioMerieux SA, Chemin de l'Orme, Marcy l'Etoile, France.
Abstract
Dengue virus (DV) infection is the most common mosquito-born viral disease of public health significance. Though most patients only suffer from flu-like symptoms, a small group of patients experiences more severe forms of the disease. The viral nonstructural protein 1 (NS1), a secreted protein correlating with viremia, is a key element used for dengue diagnosis with potential implications in severe dengue prognosis. Capture-ELISAs for the early detection of the NS1protein in the sera during the acute febrile stage are commonly used in routine by diagnostic laboratories. In this study, the detection of NS1 protein in DV-infected material was assessed by an alternative method combining a single NS1-directed monoclonal antibody and the SELDI-TOF/MS technology. According to the epitope mapping, the antibodies used are mainly directed against an immuno-dominant peptide located on the C-terminal part of the protein. The NS1 SELDI-TOF assay is specific, has a sensitivity level close to capture-ELISAs and is potentially useful for a coupled serotyping/detection assay or for the detection of subtle post-translational modifications on the protein. J. Med. Virol. 84:490-499, 2012. © 2011 Wiley Periodicals, Inc.
Copyright © 2012 Wiley Periodicals, Inc.
- PMID:
- 22246837
- [PubMed - in process]
Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates.
Source
Instituto de la Grasa-CSIC, Av. Padre García Tejero, 4, 41012-Seville, Spain. mdmar@cica.es.
Abstract
BACKGROUND:
Some dietary proteins possess biological properties which make them potential ingredients of functional or health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme.
RESULTS:
All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea proteinisolate. The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by 30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: β-carotene bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant activity in all assays, especially reducing power and DPPH scavenging effect than chickpeaprotein isolate.
CONCLUSION:
The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients. The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time. Copyright © 2012 Society of Chemical Industry.
Copyright © 2012 Society of Chemical Industry.
- PMID:
- 22246802
- [PubMed - as supplied by publisher]
Mass Spectrometry-based Proteomics Strategies for Protease Cleavage Sites Identification.
Source
AG Systematische Proteomforschung, Institut für Experimentelle Medizin, Christian-Albrechts-Universität, Kiel, Germany. b.vdberg@iem.uni-kiel.de.
Abstract
Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal posttranslational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, the understanding of the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatics tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches addressed. Recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22246699
- [PubMed - as supplied by publisher]
Protective effect of Melothria maderaspatana leaf fraction on electrolytes, catecholamines, endothelial nitric oxide synthase and endothelin-1 peptide in uninephrectomized deoxycorticosterone acetate-salt hypertensive rats.
Source
Department of Community Health Sciences, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh, 11433, Saudi Arabia.
Abstract
This study was designed to investigate the protective effect of ethyl acetate fraction of Melothria maderaspatana (EAFM) leaf on electrolytes, catecholamines, endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) peptide in uninephrectomized deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Administration of DOCA-salt significantly increased the systolic and diastolic blood pressure and treatment with EAFM significantly lowered the blood pressure. In DOCA-salt rats, the levels of sodium and chloride increased significantly while potassium level decreased and administration of EAFM brought these parameters to normality. The levels of epinephrine and norepinephrine increased significantly in DOCA-salt rats and administration of EAFM significantly decreased these parameters to normality. DOCA-salt hypertensive rats exhibited significantly decreased L: -arginine and nitrite + nitrate levels and administration of EAFM brought these parameters to normality. DOA-salt hypertensive rats showed down-regulation of eNOS and up-regulation of ET-1 protein expressions in heart and kidney, and treatment with EAFM prevented down-regulation of eNOS and significantly down-regulated the ET-1 protein expressions. In conclusion, EAFM provides good blood pressure control by enhancing potassium and decreasing sodium levels, decreasing levels of epinephrine and norepinephrine, and preventing down-regulation of eNOS and significantly down-regulating ET-1 proteinexpression.
- PMID:
- 22246664
- [PubMed - as supplied by publisher]
Neuroplasticity of sensory and sympathetic nerve fibers in the painful arthritic joint.
Source
Research Service, VA Medical Center, One Veterans Drive, Minneapolis, MN 55417, USA.
Abstract
OBJECTIVE.: Many forms of arthritis are accompanied by significant chronic joint pain. Here we studied whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathological reorganization. METHODS.: A painful arthritic knee joint was produced by injection of complete Freund's adjuvant (CFA) into the knee joint of young adult mice. CFA-injected mice were then treated systemically with vehicle or anti-NGF antibody. Pain behaviors were assessed and at 28 days following the initial CFA injection, the knee joints were processed for immunohistochemistry using antibodies raised against calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kDa (NF200; sensory nerve fibers), growth associated protein-43 (GAP43; sprouted nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), CD31 (endothelial cells) or CD68 (monocytes/macrophages). RESULTS.: In CFA-injected mice, but not vehicle-injected mice, there was a significant increase in the density of CD68(+) macrophages, CD31(+) blood vessels, CGRP(+) , NF200(+) , GAP43(+) , and TH(+) nerve fibers in the synovium as well as joint pain-related behaviors. Administration of anti-NGF reduced these pain-related behaviors and the ectopic sprouting of nerve fibers, but had no significant effect on the increase in density of CD31(+) blood vessels or CD68(+) macrophages. CONCLUSIONS.: Ectopic sprouting of sensory and sympathetic nerve fibers occurs in the painful arthritic joint and may be involved in the generation and maintenance of arthritic pain.
Copyright © 2012 by the American College of Rheumatology.
- PMID:
- 22246649
- [PubMed - as supplied by publisher]
mTORC1 inhibition and ECM-cell adhesion-independent drug resistance via PI3K-AKT and PI3K-RAS-MAPK feedback loops.
Source
Miller School of Medicine, University of Miami Health System, 1600 NW 10th Avenue, Suite 8006 (R-2), Miami, FL, 33136, USA, kgaloian@med.miami.edu.
Abstract
Mammalian target of rapamycin (mTOR) serine threonine kinase is the enzyme that regulates cancer cell growth by altering nutrient supplies to cancer cells. The neuropeptide (proline-rich peptide 1 (PRP-1)), galarmin, produced by the brain neurosecretory cells is a mTOR kinase inhibitor with powerful 80% antiproliferative cytostatic effect in a high-grade chondosarcoma and other mesenchymal tumors. However, the negative feedback loop of phosphatidylinositol 3 kinase-Protein kinase B (PKB), PI3K-AKT and PI3K-rat sarcoma (RAS)-mitogen-activated protein kinase (MAPK) activation is well documented for mTOR inhibitors. This study explored the involvement of those loops in drug resistance after the treatment with mTOR complex 1 (mTORC1) inhibitor, PRP-1. Multidrug resistance assay (MDR) demonstrated that this cytokine did not inhibit permeability glycoprotein-mediated MDR in chondrosarcoma. Phospho-MAPK array in human chondrosarcoma cell line treated with galarmin (10 μg/ml,) showed a strong upregulation of phosphorylated glycogen synthase kinase 3β (GSK3β) via activation of PI3K-AKT and MAPK feedback loops. Such GSK3β inactivation leads to β-catenin accumulation that entails drug resistance. The ability of cells to metastasize is reflected in their capacity to adhere to extracellular matrix and endothelium. Laminin cell adhesion assay demonstrated that PRP-1 in the same concentrations that inhibit mTOR kinase inhibited JJ012 chondrosarcoma cell adhesion. The neuropeptide did not have any effect on the expression of total focal adhesion kinase and its phosphorylated form. Thus, it was not accompanied by total HAT downregulation and total HDAC upregulation. Combinatorial treatments of PRP-1 with MAPK and PI3K/AKT inhibitors most probably will lead to full cytotoxicity overcoming drug resistance.
- PMID:
- 22246604
- [PubMed - as supplied by publisher]
Study on the chaperone properties of conserved GTPases.
Source
RNA Lab, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
Abstract
As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascentpeptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.
- PMID:
- 22246579
- [PubMed - as supplied by publisher]
One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes.
Source
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114, Wrocław, Poland.
Abstract
Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.
- PMID:
- 22246380
- [PubMed - as supplied by publisher]
Full-length structural model of RET3 and SEC21 in COPI: identification of binding sites on the appendage for accessory protein recruitment motifs.
Source
Department of Anatomy and Cell Biology, McGill University, Strathcona Building, 3640 University Street, Montreal, QC H3A 2B2, Canada.
Abstract
COPI, a 600 kD heptameric complex (consisting of subunits α, β, γ, δ, ε, ζ, and β') "coatomer," assembles non-clathrin-coated vesicles and is responsible for intra-Golgi and Golgi-to-ER protein trafficking. Here, we report the three-dimensional structures of the entire sequences of yeast Sec21 (γ-COPI mammalian ortholog), yeast Ret3 (ζ-COPI mammalian ortholog), and the results of successive molecular dynamics investigations of the subunits and assembly based on a protein-protein docking experiment. The three-dimensional structures of the subunits in their complexes indicate the residues of the two subunits that impact on assembly, the conformations of Ret3 and Sec21, and their binding orientations in the complexed state. The structure of the appendage domain of Sec21, with its two subdomains-the platform and the β-sandwich, was investigated to explore its capacity to bind to accessory protein recruitment motifs. Our study shows that a binding site on the platform is capable of binding the Eps15 DPF and epsin DPW2peptides, whereas the second site on the platform and the site on the β-sandwich subdomain were found to selectively bind to the amphiphysin FXDXF and epsin DPW1 peptides, respectively. Identifying the regions of both the platform and sandwich subdomains involved in binding each peptide motif clarifies the mechanism through which the appendage domain of Sec21 engages with the accessory proteins during the trafficking process of non-clathrin-coated vesicles.
- PMID:
- 22246286
- [PubMed - as supplied by publisher]
Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog.
Source
Micropharma Ltd, 141 President Kennedy Avenue, Université de Quebec à Montreal (UQAM), Biological Sciences Building Unit 5569, Montreal, QC, Canada, H2X 3Y7.
Abstract
The terrestrial anuran Rana sylvatica tolerates extended periods of whole-body freezing during the winter. Freezing survival is facilitated by extensive glycogen hydrolysis and distribution of high concentrations of the cryoprotectant glucose into blood and all tissues. As glycogenesis is both an energy-expensive process and counter-productive to maintaining sustained high cryoprotectant levels, we proposed that glycogen synthase kinase-3 (GSK-3) would be activated when wood frogs froze and would phosphorylate its downstream substrates to inactivate glycogen synthesis. Western blot analysis determined that the amount of phosphorylated (inactive) GSK-3 decreased in all five tissues tested in 24 h frozen frogs compared with unfrozen controls. Total GSK-3 protein levels did not change, with the exception of heart GSK-3, indicating that post-translational modification was the primary regulatory mechanism for this kinase. Kinetic properties of skeletal muscle GSK-3 from control and frozen frogs displayed differential responses to a temperature change (22 versus 4°C) and high glucose. For example, when assayed at 4°C, the K(m) for the GSK-3 substrate peptide was ∼44% lower for frozen frogs than the corresponding value in control frogs, indicating greater GSK-3 affinity for its substrates in the frozen state. This indicates that at temperatures similar to the environment encountered by frogs, GSK-3 in frozen frogs will phosphorylate its downstream targets more readily than in unfrozen controls. GSK-3 from skeletal muscle of control frogs was also allosterically regulated. AMP and phosphoenolpyruvate activated GSK-3 whereas inhibitors included glucose, glucose 6-phosphate, pyruvate, ATP, glutamate, glutamine, glycerol, NH(4)Cl, NaCl and KCl. The combination of phosphorylation and allosteric control argues for a regulatory role of GSK-3 in inactivating glycogenesis to preserve high glucose cryoprotectant levels throughout each freezing bout.
- PMID:
- 22246263
- [PubMed - as supplied by publisher]
Mitochondria-targeted antioxidant attenuates high glucose-induced P38 MAPK pathway activation in human neuroblastoma cells.
Source
Department of Endocrinology Provincial Hospital Affiliated to Shandong University, Jinan 250021, P.R. China.
Abstract
Excessive mitochondrial free radical production and the related mitogen-activated protein kinase P38 (P38 MAPK) activation are key regulators in the pathogenesis of high glucose-induced cell stress. Increasing evidence has emphasized the impact of hyperglycemia on neurons and the consequent neuronal stresses eventually resulting in neurodegeneration and neuronal death. In this study, we employed a novel mitochondria-targeted antioxidant, SS31peptide, on high glucose-insulted neuroblastoma cells (SH-SY5Y). Our results showed that high glucose promoted significantly increased P38 phosphorylation which was efficiently suppressed by the application of the SS31 peptideunder the experimental conditions. The inhibition of high glucose-induced P38 activation by the SS31 peptide was associated with the impact of the SS31 peptide on attenuating high glucose-induced mitochondrial ROS (reactive oxygen species) elevation and mitochondrial membrane potential collapse. The addition of SS31 peptide significantly attenuated high-gluose-induced apoptosis. Therefore, our study suggests that elimination of high glucose-induced mitochondrial oxidative stress helps to rescue SH-SY5Y cells from high glucose-related P38 MAPK pathway disturbances, and the SS31 peptide has the potential to serve as a new treatment strategy against hyperglycemia-instigated neuronal perturbations.
- PMID:
- 22245807
- [PubMed - as supplied by publisher]
Immobilizing CC chemokine receptor 4's N-terminal extracellular tail on a capillary to study its potential ligands by capillary electrophoresis.
Source
Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, People's Republic of China.
Abstract
ML40 is the equivalent peptide derived from the N terminal of CCC4 (CC chemokine receptor 4), which plays a pivotal role in allergic inflammation. A new capillary electrophoresis method was developed to study the interactions between ML40 and its potential ligands in which ML40 was immobilized on the inner wall of capillary as the stationary phase based on the covalent linking technique. The interaction between S009, a known CCR4 antagonist, and the immobilized ML40 was studied to validate the bioactivity of ML40. The electropherogram of S009 showed that the peak height was reduced and the peak width was broadened in the ML40 immobilized capillary. Otherwise, 25 computer-aided design and drafting compounds were screened out using this method. Four compounds' peak widths were broadened and their peak heights were reduced, as with S009. Meanwhile, nonlinear chromatography was used to calculate the constants for the ligand-receptor complex formation. Furthermore, the tertiary amine compounds belonging to the chiral tertiary amines of the type NRR'R″, which are optically inactive resulting from rapid pyramide inversion, were chiral separated by our protein immobilization method for the first time. In general, the methodology presented would be applicable to study compound-ML40 interactions as a reliable and robust screening method for CCR4 antagonist discovery.
Copyright © 2012. Published by Elsevier Inc.
- PMID:
- 22245764
- [PubMed - as supplied by publisher]
A novel molluscan sigma-like glutathione S-transferase from Manila clam, Ruditapes philippinarum: Cloning, characterization and transcriptional profiling.
Source
Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Special Self-Governing Province, 690-756, Republic of Korea.
Abstract
Glutathione S-transferases (GSTs) are versatile enzymes, act as primary intracellular detoxifiers and contribute to a broad range of physiological processes including cellular defense. In this study, a full-length cDNA representing a novel sigma-like GST was identified from Manila clam, Ruditapes philippinarum (RpGSTσ). RpGSTσ (884bp) was found to possess an open reading frame of 609bp. The encoded polypeptide (203 amino acids) had a predicted molecular mass of 23.21kDa and an isoelectric point of 7.64. Sequence analysis revealed two conserved GST domain profiles in N- and C-termini. Alignment studies revealed that the identity between deduced peptides of RpGSTσ and known GSTσ members was relatively low (<35%), except a previously identified Manila clam GSTσ isoform (87.2%). Phylogenetic analysis indicated that RpGSTσ clustered together with molluscan GSTσ homologs, which were closely related to insect GSTσs. The RpGSTσ was subsequently cloned and expressed as recombinant protein, in order to characterize its biological activity. The recombinant RpGSTσ exhibited characteristic glutathione conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene and ethacrynic acid. It had an optimal pH and temperature of 8.0 and 35°C, respectively. Expression profiles under normal conditions and in response to lipopolysaccharide-, poly I:C- and Vibrio tapetis-challenges were also investigated. RpGSTσ demonstrated a differential tissue distribution with robust transcription in gills of normal animals. We explored potential association of GSTσ in cellular defense during bacterial infection and found that in challenged clams, RpGSTσ gene was significantly induced in internal and external tissues, in conjunction with manganese- as well as copper-zinc superoxide dismutase (MnSOD and CuZnSOD) genes. Moreover, the induction was remarkably higher in hemocytes than in gill. Collectively, our findings suggested that RpGSTσ could play a significant role in cellular defense against oxidative stress caused by bacteria, in conjunction with other antioxidant enzymes, such as SODs.
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