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The Effects of the Citrullinating Enzyme, Peptidylarginine Deiminase, on the Activation of T Cells
The effects of the citrullinating enzyme, peptidylarginine deiminase, on the activation of T cells Rita Barreto Duarte Carilho Torrão Doctor of Philosophy Aston University September, 2016 © Rita Barreto Duarte Carilho Torrão, 2016 Rita Barreto Duarte Carilho Torrão asserts her moral right to be identified as the author of this thesis. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the report and no information derived from it may be published without proper acknowledgement. 1 Aston University The effects of the citrullinating enzyme, peptidylarginine deiminase, on the activation of T cells Rita Barreto Duarte Carilho Torrão Doctor of Philosophy 2016 Rheumatoid arthritis (RA) and periodontitis (PID) are two chronic inflammatory diseases associated with the modification of self-proteins by citrullinating peptidyl arginine deiminase (PAD) enzymes, leading to a loss of tolerance by the immune system. The main goal of this study was to explore the action of PAD enzyme- mediated citrullination on T cell membrane proteins and gene expression in relation to the T cell phenotype in PID. Effects on cells of the adaptive immune system have been less well studied in PID and the data obtained here shows that citrullination of peripheral blood mononuclear cells (PBMC) by PAD enzymes impairs T cell activation. Microarray studies showed that PAD enzyme treatment led to the dysregulation of genes involved in glucose and amino acid metabolism in PBMC. Real time quantitative polymerase chain reaction (RT-QPCR) in CD4 and CD8 T cells from PID patients showed a trend towards down-regulation of hexokinase 3 and up-regulation of argininosuccinate synthase1. -
The Intrinsically Disordered Proteins of Myelin in Health and Disease
cells Review Flexible Players within the Sheaths: The Intrinsically Disordered Proteins of Myelin in Health and Disease Arne Raasakka 1 and Petri Kursula 1,2,* 1 Department of Biomedicine, University of Bergen, Jonas Lies vei 91, NO-5009 Bergen, Norway; [email protected] 2 Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Aapistie 7A, FI-90220 Oulu, Finland * Correspondence: [email protected] Received: 30 January 2020; Accepted: 16 February 2020; Published: 18 February 2020 Abstract: Myelin ensheathes selected axonal segments within the nervous system, resulting primarily in nerve impulse acceleration, as well as mechanical and trophic support for neurons. In the central and peripheral nervous systems, various proteins that contribute to the formation and stability of myelin are present, which also harbor pathophysiological roles in myelin disease. Many myelin proteins have common attributes, including small size, hydrophobic segments, multifunctionality, longevity, and regions of intrinsic disorder. With recent advances in protein biophysical characterization and bioinformatics, it has become evident that intrinsically disordered proteins (IDPs) are abundant in myelin, and their flexible nature enables multifunctionality. Here, we review known myelin IDPs, their conservation, molecular characteristics and functions, and their disease relevance, along with open questions and speculations. We place emphasis on classifying the molecular details of IDPs in myelin, and we correlate these with their various functions, including susceptibility to post-translational modifications, function in protein–protein and protein–membrane interactions, as well as their role as extended entropic chains. We discuss how myelin pathology can relate to IDPs and which molecular factors are potentially involved. Keywords: myelin; intrinsically disordered protein; multiple sclerosis; peripheral neuropathies; myelination; protein folding; protein–membrane interaction; protein–protein interaction 1. -
Citrullination of CXCL8 by Peptidylarginine Deiminase Alters
ARTICLE Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue infl ammation Paul Proost , 1 Tamara Loos , 1 Anneleen Mortier , 1 Evemie Schutyser , 1 Mieke Gouwy , 1 Samuel Noppen , 1 Chris Dillen , 2 Isabelle Ronsse , 1 Ren é Conings , 1 Sofi e Struyf , 1 Ghislain Opdenakker , 2 Prabhat C. Maudgal , 3 and Jo Van Damme 1 1 Laboratory of Molecular Immunology and 2 Laboratory of Immunobiology, Rega Institute, 3 Laboratory of Ophthalmology, University Hospital, K.U.Leuven, B 3000 Leuven, Belgium Biological functions of proteins are infl uenced by posttranslational modifi cations such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttrans- lational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1 – 77), generating CXCL8(1 – 77)Cit5 . Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD effi ciently and site- specifi cally citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1 . In comparison with CXCL8(1 – 77), CXCL8(1 – 77)Cit5 had reduced affi nity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1– 77), CXCL8(1 – 77)Cit5 was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6– 77). Upon intraperitoneal injection, CXCL8(6 – 77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1 – 77). -
Ryanodine Receptors Are Part of the Myospryn Complex in Cardiac Muscle Received: 10 March 2017 Matthew A
www.nature.com/scientificreports OPEN Ryanodine receptors are part of the myospryn complex in cardiac muscle Received: 10 March 2017 Matthew A. Benson1, Caroline L. Tinsley2, Adrian J. Waite 2, Francesca A. Carlisle2, Steve M. Accepted: 12 June 2017 M. Sweet3, Elisabeth Ehler4, Christopher H. George5, F. Anthony Lai5,6, Enca Martin-Rendon7 Published online: 24 July 2017 & Derek J. Blake 2 The Cardiomyopathy–associated gene 5 (Cmya5) encodes myospryn, a large tripartite motif (TRIM)- related protein found predominantly in cardiac and skeletal muscle. Cmya5 is an expression biomarker for a number of diseases afecting striated muscle and may also be a schizophrenia risk gene. To further understand the function of myospryn in striated muscle, we searched for additional myospryn paralogs. Here we identify a novel muscle-expressed TRIM-related protein minispryn, encoded by Fsd2, that has extensive sequence similarity with the C-terminus of myospryn. Cmya5 and Fsd2 appear to have originated by a chromosomal duplication and are found within evolutionarily-conserved gene clusters on diferent chromosomes. Using immunoafnity purifcation and mass spectrometry we show that minispryn co-purifes with myospryn and the major cardiac ryanodine receptor (RyR2) from heart. Accordingly, myospryn, minispryn and RyR2 co-localise at the junctional sarcoplasmic reticulum of isolated cardiomyocytes. Myospryn redistributes RyR2 into clusters when co-expressed in heterologous cells whereas minispryn lacks this activity. Together these data suggest a novel role for the myospryn complex in the assembly of ryanodine receptor clusters in striated muscle. Te unique cytoskeletal organisation of striated muscle is dependent upon the formation of specialised interac- tions between proteins that have both structural and signalling functions1. -
Deimination, Intermediate Filaments and Associated Proteins
International Journal of Molecular Sciences Review Deimination, Intermediate Filaments and Associated Proteins Julie Briot, Michel Simon and Marie-Claire Méchin * UDEAR, Institut National de la Santé Et de la Recherche Médicale, Université Toulouse III Paul Sabatier, Université Fédérale de Toulouse Midi-Pyrénées, U1056, 31059 Toulouse, France; [email protected] (J.B.); [email protected] (M.S.) * Correspondence: [email protected]; Tel.: +33-5-6115-8425 Received: 27 October 2020; Accepted: 16 November 2020; Published: 19 November 2020 Abstract: Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes. Keywords: citrullination; post-translational modification; cytoskeleton; keratin; filaggrin; peptidylarginine deiminase 1. Introduction Intermediate filaments (IF) constitute a unique macromolecular structure with a diameter (10 nm) intermediate between those of actin microfilaments (6 nm) and microtubules (25 nm). In humans, IF are found in all cell types and organize themselves into a complex network. They play an important role in the morphology of a cell (including the nucleus), are essential to its plasticity, its mobility, its adhesion and thus to its function. -
Dystrophin Complex Functions As a Scaffold for Signalling Proteins☆
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1838 (2014) 635–642 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Review Dystrophin complex functions as a scaffold for signalling proteins☆ Bruno Constantin IPBC, CNRS/Université de Poitiers, FRE 3511, 1 rue Georges Bonnet, PBS, 86022 Poitiers, France article info abstract Article history: Dystrophin is a 427 kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and Received 27 May 2013 incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex Received in revised form 22 August 2013 (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins Accepted 28 August 2013 and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force trans- Available online 7 September 2013 duction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaf- fold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling Keywords: Dystrophin-associated protein complex (DAPC) processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. syntrophin The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle Sodium channel cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More TRPC channel generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. -
Adhesive Properties and Inflammatory Potential of Citrullinated Myelin
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Neurochem Res (2012) 37:1959–1966 DOI 10.1007/s11064-012-0816-z ORIGINAL PAPER Adhesive Properties and Inflammatory Potential of Citrullinated Myelin Basic Protein Peptide 45–89 Lali V. Shanshiashvili • Irina V. Kalandadze • Jeremy J. Ramsden • David G. Mikeladze Received: 12 July 2011 / Revised: 21 May 2012 / Accepted: 26 May 2012 / Published online: 8 June 2012 Ó Springer Science+Business Media, LLC 2012 Abstract Deimination of arginyl residue of myelin basic of the transcription factor NF-kB in these processes. Our protein (MBP) reduces cationicity of MBP and impedes the results suggest that some citrullinated peptides, initially normal myelin membrane assembly. Less ordered structure released from oligodendrocytes, might activate microglia, of MBP is more susceptible to proteolytic attack that may which produces reactive nitrogen species and generates in lead to the release of highly immunogenic deiminated turn fatal feedbacks that kill oligodendrocytes. peptides into extracellular milieu. We have studied the association of peptides 45–89 derived from citrullinated Keywords Myelin basic protein Á Deimination Á MBP (C8 isomer) and phosphorylated MBP (C3 isomer) Citrullinated peptides Á Myelin lipids Á Primary Glial cells Á with the myelin lipids in a model membrane system using Inflammation optical waveguide lightmode spectrometry. The analysis of association/dissociation kinetics to planar lipids under controlled hydrodynamic conditions has shown that MBP Introduction 45–89 peptide from citrullinated C8 isomer is less effec- tively adsorbed on the lipid membrane, than peptide from Activated microglia is significant component of the brain phosphorylated C3 isomer and packing densities for pathology during the chronic neuroinflammatory diseases. -
Citrullination: Taking the Charge out Of
CYTOSKELETON NEWS NEWS FROM CYTOSKELETON INC. this issue Citrullination: Taking the Charge out of Arg Related Publications Oct Research Tools 2014 Meetings Citrullination: Taking the Charge out of Arg Society for Neuroscience Protein citrullination (a.k.a. deimination) is a novel arginine- RA, several proteins have been identified that are specifically 2014 directed post-translational modification (PTM) that results in citrullinated in the synovial fluid of arthritic joints10; many of News November 15-19, a permanent change in the targeted protein. Peptidylarginine which are mentioned above. The citrullination of these proteins Washingon, D.C. deiminases (PADs) mediate the calcium-dependent deimination results in novel epitopes that give rise to autoantibodies7, and Booth #1917 of the guanidino group of arginine side chains to form an ureido the resulting anti-citrullinated protein antibodies (ACPAs) have group and the nonstandard amino acid citrulline (see Fig. 1). become a standard diagnostic and prognostic indicator for RA15- ASCB/IFCB 2014 There are 5 different PAD isoforms (PAD1-4, PAD6) that share 17. Circulating ACPAs are often present before other symptoms of December 6-10, significant sequence homology and differ primarily in their tissue- RA and they are associated with an earlier onset of the disease, Philadelphia, PA specific expression1. PADs are incapable of deiminating free more severe joint damage, and a higher risk of cardiovascular co- Booth #818 L-arginine, which confirms their primary role in the modification morbidities15-17. of arginine side chains present in proteins2. To date, there have been no enzymes identified that can reverse this process. Cytoskeleton Publications Products The deimination of arginine side chains in proteins results in the net loss of a positive charge and an increase in local hydrophobicity Actin Proteins for the target protein. -
The Neurotransmitter Released at the Neuromuscular Junction Is
The Neurotransmitter Released At The Neuromuscular Junction Is Towney congeals his bibbers manipulate jocular, but cash-and-carry Winfred never extravasating so malcontentedly. Is Norton always dipetalous and unworn when guzzled some admeasurement very untidily and adversely? Busty Dominic overexcited some close and jemmying his galluses so nautically! Hiw are four nlgs are working memory, sv hubs depending on the specific to the neurotransmitter released neuromuscular junction at institutions across brain The energy is delivered in a fractional manner. Smooth muscle NMJ is formed between the autonomic nerve fibers that branch diffusely on strength muscle in form diffuse junctions. In an intact brain, volume was observed that Cac density at AZs is indeed strongly correlated with Pr. Chemical synapses involve the transmission of chemical information from one cell as the next. Currents through the fusion pore that forms during exocytosis of a secretory vesicle. If you order something abusive or that does not lessen with surrender terms or guidelines please flag it as inappropriate. Ach in the presynaptic protein in the active secretors of the released. You can login by using one alongside your existing accounts. Many drugs and anesthetic agents also affect neuromuscular junction and impulse transmission to inside their effects. The chemical must be present within a neuron. Another route for tetanus is lockjaw, respectively, the calcium rushes out of newly opened gates. In our data presented at multiple neurotransmitters are commonly performed by abnormal nmj but the neurotransmitter released at is the neuromuscular junction, it will fail to propagate from another power stroke can have qualitatively distinct categories reflective of the resultant of development. -
Evolution of a Mass Spectrometry-Grade Protease with PTM-Directed Specificity
Evolution of a mass spectrometry-grade protease with PTM-directed specificity Duc T. Trana, Valerie J. Cavetta, Vuong Q. Danga, Héctor L. Torresa, and Brian M. Paegela,1 aDepartment of Chemistry, The Scripps Research Institute, Jupiter, FL 33458 Edited by David Baker, University of Washington, Seattle, WA, and approved November 8, 2016 (received for review July 7, 2016) Mapping posttranslational modifications (PTMs), which diversely The relationship between protein cleavage and MS sequence modulate biological functions, represents a significant analytical coverage has spurred the exploration of methods to induce alternate challenge. The centerpiece technology for PTM site identification, cleavages, especially at PTM sites. Examples include chemoselective mass spectrometry (MS), requires proteolytic cleavage in the vicinity of pSer side chain modification to establish pSer-dependent tryptic a PTM to yield peptides for sequencing. This requirement catalyzed our cleavage sites (7, 8), and directed evolution of novel proteolytic efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a cleavage activity, yielding a pTyr-dependent subtilisin mutant (9) and PTM implicated in epigenetic and immunological function, made an a suite of OmpT mutants that cleaved the Ala–Arg (10), sTyr–Arg ideal first target, because citrullination eliminates arginyl tryptic sites. (11), or nTyr–Arg (12) P1–P1′ peptide bonds; P1–P1′ cleavage Bead-displayed trypsin mutant genes were translated in droplets, the junction requirements precluded further implementation. Proteo- mutant proteases were challenged to cleave bead-bound fluorogenic lytic specificity has also been successfully evolved in other enzymes probes of citrulline-dependent proteolysis, and the resultant beads (13, 14), but the MS workhorse enzyme, trypsin, has remained un- (1.3 million) were screened. -
Label Value Core Facility Name Mass Spectrometry and Proteomics Facility Last Name Robert First Name Cole
Label Value Core Facility Name Mass Spectrometry and Proteomics Facility Last Name Robert First Name Cole Email [email protected] Phone 410-614-6968 Amount of Funding $25000 Requested Briefly describe the core The Johns Hopkins University School of Medicine Mass Spectrometry and Proteomics services you offer: Facility assists investigators with identifying and quantifying proteins and their modifications that are deferentially expressed in cells, tissues or body fluids, with tracking interactions with binding partners during changes in signal transduction, and with identifying proteolytic cleavage sites and mapping post-translational modifications by providing the following proteomic services: Consultation: The Core Director, Core Proteomics Specialist and, when appropriate, a Biostatistician, will have pre- and post-analysis discussions with investigators on project goals, experimental design, sample preparation procedures and data analysis as required for successful mass spectrometry analysis. Sample preparation: The Core staff will advise, teach or assist investigators’ students and fellows in the current protein extraction and sample preparation techniques, including buffer exchange, column chromatography, proteolytic digestion and stable isotope labeling, to ensure high quality, reproducibility and continuity of sample preparation. Protein Identification: The Core will identify and characterize proteins from complex mixtures of proteins in solution, in gel bands or spots, by liquid chromatography interfaced with tandem MS (LCMS/MS) using the single or multi-dimensional protein identification technology (MuDPIT). Investigators will receive an interactive results file containing all proteins identified, are taught how to navigate through their data and are advised on methods to verify the presence of identified proteins. Protein Modifications: The Core staff will perform or train investigators to enrich samples for modified peptides using chemical (e.g.TiO2 for Ser/Thr/Tyr phosphorylated peptides) or immunoprecipitation methods (e.g. -
Citrullination a Potential Post-Translational Modification Linked to TDP-43 Pathology Patricia Rocha-Rangel1, Zainuddin Quadri1, Dale Chaput3, Daniel C
Citrullination a potential post-translational modification linked to TDP-43 pathology Patricia Rocha-Rangel1, Zainuddin Quadri1, Dale Chaput3, Daniel C. Lee PhD2, Maj-Linda B. Selenica PhD1 1Sanders Brown Center on Aging, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, United States; 2Sanders Brown Center on Aging, Department of Neuroscience, University of Kentucky, Lexington, KY, United States; 3Proteomics and Mass Spectrometry Core Facility, Florida Center of Excellence for Drug Discovery and Innovation (CDDI), University of South Florida, 3720 Spectrum Blvd, Suite 303, Tampa, FL 33612, USA. Induced Citrullinated TDP-43 levels in Tar transgenic INTRODUCTION OBJECTIVES animal model TDP-43 is a nuclear RNA/DNA binding protein that in its pathological form, . Investigate PAD4 expression and activity in neurons of TAR transgenic A. Non-Tg TAR4 TAR4/4 mislocalizes and aggregates into the cytoplasm as insoluble cellular mice citR83 inclusions. TDP-43 inclusions are the histological hallmarks of . Evaluate the affinity of different CitTDP-43R antibodies during pathology 50µm frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and progression in TAR mouse models amyotrophic lateral sclerosis (ALS). Currently, mechanisms responsible for . Determine the CitTDP-43R protein structure in the neurons of TAR 50µm TDP-43 mislocalization and aggregation remain unclear, but it is transgenic mice hypothesized that post translational modifications (PTMs) play an active citR268/272 role. Citrullination is an irreversible PTM in which peptidyl arginine Identification of Citrullinated TDP-43 by mass 50µm deiminases (PADs) catalyze the conversion of arginine to citrulline. Little is spectrometry known about PADs in neurodegeneration, but there is few evidence of 50µm increased PAD4 expression in Alzheimer’s Disease (AD) and ALS motor A.