DOCSLIB.ORG

Protein TAILS: When Termini Tell Tales of Proteolysis and Function

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Protein TAILS: When Termini Tell Tales of Proteolysis and Function Available online at www.sciencedirect.com Protein TAILS: when termini tell tales of proteolysis and function 1,2,3 1,2,3 Philipp F Lange and Christopher M Overall Among the hundreds of posttranslational modifications, limited functions of bioactive proteins will be opaque and hence proteolysis, also known as processing, is special: It is hypotheses based on traditional shotgun analyses, may be irreversible, near ubiquitous, and by trimming peptide chains misleading or even worse, totally wrong. from their ends or cutting proteins into two, proteolysis forms shorter chains displaying new termini. The unique chemistry PTM of proteins constitutes a highly diverse and dynamic and location of a-amino-termini and carboxyl-termini in a regulatory layer affecting all aspects of a protein from protein engender special chemical and physical properties to a protein folding, localization, interaction and bioactivity protein. Hence, modification of protein termini is often to its stability and ultimately degradation. Therefore, each associated with new biological activities of a protein. We distinctly modified version of a protein, also called a protein highlight recent proteomic developments enabling high species, and not just the initial translated version, needs to throughput identification of protein termini. This has be considered as the functional units comprising the pro- revolutionized degradomics and protein characterization by teome [3]. The diversity of reversible and irreversible mapping the specificity of terminal modifications and of modifications as well as the extensive modification machin- proteases, and has been used to directly identify new protease ery [4] and the possibility of combinatorial effects substrates and molecular pathways altered by proteolysis. dramatically increase proteome complexity by several Addresses orders. Organisms as different as worm, fly and man have 1 University of British Columbia, Department of Oral Biological and comparable sized genomes yet show a great discrepancy in Medical Sciences, 4.401 Life Sciences Institute, 2350 Health Sciences phenotypic complexity. While splicing introduces bulk Mall, Vancouver, BC, Canada V6T 1Z3 2 complexity it might well be that the diversity created by University of British Columbia, Department of Biochemistry and Molecular Biology, 4.401 Life Sciences Institute, 2350 Health Sciences pinpoint posttranslational modifications accounts for the Mall, Vancouver, BC, Canada V6T 1Z3 observed phenotypic differences. Hence, advanced pro- 3 University of British Columbia, Centre for Blood Research, 4.401 Life teomics has potential to explain phenotypes where con- Sciences Institute, 2350 Health Sciences Mall, Vancouver, BC, Canada ventional genomics fall short — but it is not easy. V6T 1Z3 Corresponding author: Overall, Christopher M ([email protected]) Every modification adds to the functional diversity of the proteome by reversibly or irreversibly converting one protein species into another that potentially is a function- Current Opinion in Chemical Biology 2013, 17:73–82 ally distinct species. In this regard, limited proteolysis is This review comes from a themed issue on Omics special as it has the unique ability to irreversibly convert Edited by Matthew Bogyo and Pauline M Rudd one into two distinct protein species while at the same For a complete overview see the Issue and the Editorial time generating new protein termini serving as attach- ment sites for even further PTM. Available online 6th January 2013 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights Second only to ubiquitin ligases in number, proteases and reserved. their inhibitors constitute a large enzyme family with 567 http://dx.doi.org/10.1016/j.cbpa.2012.11.025 members in humans. In what has been termed the degradome, the assembly of all elements involved in proteolysis — proteases, inhibitors and the processed Introduction substrates — can now be specifically studied in high Proteomics has made astonishing advances in all areas throughput investigations termed degradomics [5 ]. Pro- including peptide enrichment, fractionation, mass spec- teases modify their substrates by hydrolysis of scissile trometry and data analysis — many of which are reviewed bonds releasing two peptide chains with the two amino in this issue. With mass spectrometry based proteomics it acids adjacent to the cleaved bond now becoming car- is now possible to identify more than 10,000 proteins from boxy-terminal or amino-terminal residues. Unlike most human cells [1,2]. Recent advances in the field of protein PTM attachment sites, the hydrolyzed peptide bond is post-translational modifications (PTMs) have uncovered not amenable for direct assessment. For limited proteol- their widespread occurrence and physiological relevance. ysis, termed processing, the site of modification is there- However, for comprehensive analysis of PTMs specific fore determined by identification of the ‘neo’ termini of peptide enrichment approaches and dedicated analyses the products. Consequently the degradome and termi- are required, without which PTMs are usually under- nome are mutually dependent, with the identification of sampled and overlooked, respectively. In the absence of termini adding considerable functional annotation to the functional annotation of proteins from PTMs many key proteome. www.sciencedirect.com Current Opinion in Chemical Biology 2013, 17:73–82 74 Omics Enriching for post-translational modifications based on the observation of one single peptide only. PTMs often occur at low stoichiometry and thus efficient For proteins having two or more such modifications, enrichment techniques are key for their successful and protein identification can often be made by two or more comprehensive identification. In general different chemi- different and unique peptides. However, for single cal affinities between the modified and unmodified peptides bearing a PTM, such as phosphopeptides, species are utilized for differential binding to a resin or unambiguous protein identification is problematic. chromatographic media yielding positive or negative For the identification of protein termini we and others selectivity and enrichment. All approaches share the introduced high confidence protein identification from common hurdle of unspecific carryover and loss following single peptide identifications based on multiple peptide binding to surfaces. A great advance for the enrichment of variants [7,8]. In the past ten years since its introduc- peptides bearing PTMs is the replacement of resins by tion [5] degradomics and its subfield, terminomics, have soluble hyper branched polyglycerol polymers leading to developed from a small field covered by only a few massively decreased nonspecific binding while increasing publications a year to a vibrant community publishing binding capacity [6 ]. over 40 papers in 2011 (Figure 1). For in depth com- parison of available mass spectrometry based methods Upon successful peptide enrichment mass spectrom- for the proteome-wide analysis of limited proteolysis etry is used for peptide and PTM identification. Unlike and their subsequent modification we refer to a recent identification of the entire protein by multiple peptides review by Huesgen and Overall [9 ] and by the in one shotgun experiment, identification of a specific accompanying paper in this issue from the Gaevert modification and often the protein bearing the PTM, is laboratory [10]. Figure 1 (a) •cell culture •lysate / secretome •cell culture •in vivo tissue + overexpressed protease + recombinant protease •in vivo cell + recombinant protease •in vivo fluid •+/+ vs. -/- 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 •Degradomics coined •ICAT protein shedding •positive selection • N-acetylation •hyperbranched •N-cyclization •cleavage kinetics •manual substrate curation polymers •negative selection for •automated •negative selection •positive selection terminal enrichment topography mapping •unbiased substrate •unbiased substrate identification identification (b)(c) Functional 50 Competence Assignment Substrate activated Identification 45 N pro Limiting peptide Complexity 40 10 N 35 P P Me Ac Cleavage Site 5 Specificity 30 10 C N 25 Terminal DAEF... Modifications ...EVKM Publications 20 Protein Termini Ac LTIS... Me MAEF... 15 Cleavage Kinetics 10 Data Integration 5 Protease Web 0 TopFIND 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Year Current Opinion in Chemical Biology Proteome-wide identification of protein termini over the decade: (a) timeline of key steps in degradomics and terminomics since its introduction. The top stream covers the type and complexity of the analytes, the bottom shows key advances. (b) Count of publications covering degradomics and terminomics over the last 10 years. (c) Summary of areas driven by advances in protein terminus identification covered in this review. Current Opinion in Chemical Biology 2013, 17:73–82 www.sciencedirect.com Protein termini and proteolysis Lange and Overall 75 Characterization of the proteolytic machinery maturation (removal of the initiator methionine, signal Since the function of a protease is inherently linked to the peptide and pro-peptide) [31 ]. More recently, in skin effect of proteolysis on its substrates, and since more than 50% of the >2000 proteins identified had evidence of half of all proteases have no annotated substrates in stable cleavage products in vivo [29 ]. MEROPS, the protease database (http://merops.sanger.- ac.uk), since 2000 a major focus has been in the identi-
Load more

Recommended publications
  • N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton
    BRIEF COMMUNICATION www.jasn.org N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton †‡ † | † Markus M. Rinschen,* § Ann-Kathrin Hoppe,* Florian Grahammer, ¶ Martin Kann,* † † † †‡ Linus A. Völker,* Eva-Maria Schurek,* Julie Binz,* Martin Höhne,* § Fatih Demir,** | †† † ‡‡ Milena Malisic,** Tobias B. Huber, ¶ Christine Kurschat,* Jayachandran N. Kizhakkedathu, †‡ †‡ Bernhard Schermer,* § Pitter F. Huesgen,** and Thomas Benzing* § *Department II of Internal Medicine, †Center for Molecular Medicine Cologne (CMMC), ‡Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), and §Systems Biology of Ageing Cologne (Sybacol), BRIEF COMMUNICATION University of Cologne, Cologne, Germany; |Department of Medicine III, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; ¶Department of Medicine IV, Medical Center and Faculty of Medicine – University of Freiburg, Freiburg, Germany; **Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany; ††BIOSS Centre for Biological Signalling Studies and Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University, Freiburg, Germany; and ‡‡Centre for Blood Research, Department of Pathology and Laboratory Medicine, Department of Chemistry, University of British Columbia, Vancouver, Canada ABSTRACT Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in of these genes leads to altered signaling maintaining the integrity of podocytes
    [Show full text]
  • Caspase Substrates and Inhibitors
    Downloaded from http://cshperspectives.cshlp.org/ on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Caspase Substrates and Inhibitors Marcin Pore˛ba1, Aleksandra Stro´z˙yk1, Guy S. Salvesen2, and Marcin Dra˛g1 1Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wrocław, Poland 2Program in Cell Death Research, Sanford-Burnham Medical Research Institute, La Jolla, California 92024 Correspondence: [email protected] Caspases are proteases at the heart of networks that govern apoptosis and inflammation. The past decade has seen huge leaps in understanding the biology and chemistry of the caspases, largely through the development of synthetic substrates and inhibitors. Such agents are used to define the role of caspases in transmitting life and death signals, in imaging caspases in situ and in vivo, and in deconvoluting the networks that govern cell behavior. Additionally, focused proteomics methods have begun to reveal the natural substrates of caspases in the thousands. Together, these chemical and proteomics technologies are setting the scene for designing and implementing control of caspase activity as appropriate targets for disease therapy. WHAT IT TAKES TO BE A CASPASE group, known as cysteine protease clan CD that he need to recycle scarce amino acids in early also contains the plant metacaspases (Vercam- Tbiotic environments required that proteases men et al. 2004), and mammalian and plant were among the first proteins to appear in the proteases of the legumain family (involved in most primitive replicating organisms. Since that specific processing events), the eukaryotic pro- distant origin, proteases have diversified into tease separase (required for sisterchromatid sep- every biological niche, occupied every cellular aration during mitosis), and the bacterial prote- and extracellular compartment, and are encod- ases clostripain and gingipain (Chen et al.
    [Show full text]
  • Full Text (PDF)
    BACK TO BASICS | PROTEASES IN LUNG DISEASE Protean proteases: at the cutting edge of lung diseases Clifford Taggart1, Marcus A. Mall2,3,4, Gilles Lalmanach5, Didier Cataldo6, Andreas Ludwig7, Sabina Janciauskiene8, Nicole Heath3,4,9, Silke Meiners10, Christopher M. Overall11, Carsten Schultz4,9, Boris Turk12 and Keren S. Borensztajn13,14 Affiliations: 1Airway Innate Immunity Research group (AiiR), Centre for Experimental Medicine, Queen’s University Belfast, UK. 2Dept of Translational Pulmonology, University of Heidelberg, Heidelberg, Germany. 3Division of Pediatric Pulmonology & Allergy and Cystic Fibrosis Center, Dept of Pediatrics, University of Heidelberg, Heidelberg, Germany. 4Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany. 5INSERM UMR1100 Centre d’Etude des Pathologies Respiratoires (CEPR), Equipe: Mécanismes Protéolytiques dans l’Inflammation, Université François Rabelais, Tours, France. 6Laboratory of Tumors and Development and Dept of Respiratory Diseases, University of Liege, Liege, Belgium. 7Inflammation Pharmacology Research Group, Institute of Pharmacology and Toxicology, RWTH Aachen University, Aachen, Germany. 8Dept of Respiratory Medicine, a member of The German Center for Lung Research (DZL), Hannover Medical School, Hannover, Germany. 9European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. 10Comprehensive Pneumology Center (CPC), University Hospital, Ludwig- Maximilians University, Helmholtz Zentrum München, Member of the German Center for
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function
    Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms
    [Show full text]
  • Biochemical Characterization and Zinc Binding Group (Zbgs) Inhibition Studies on the Catalytic Domains of Mmp7 (Cdmmp7) and Mmp16 (Cdmmp16)
    MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Fan Meng Candidate for the Degree DOCTOR OF PHILOSOPHY ______________________________________ Director Dr. Michael W. Crowder ______________________________________ Dr. David L. Tierney ______________________________________ Dr. Carole Dabney-Smith ______________________________________ Dr. Christopher A. Makaroff ______________________________________ Graduate School Representative Dr. Hai-Fei Shi ABSTRACT BIOCHEMICAL CHARACTERIZATION AND ZINC BINDING GROUP (ZBGS) INHIBITION STUDIES ON THE CATALYTIC DOMAINS OF MMP7 (CDMMP7) AND MMP16 (CDMMP16) by Fan Meng Matrix metalloproteinase 7 (MMP7/matrilysin-1) and membrane type matrix metalloproteinase 16 (MMP16/MT3-MMP) have been implicated in the progression of pathological events, such as cancer and inflammatory diseases; therefore, these two MMPs are considered as viable drug targets. In this work, we (a) provide a review of the role(s) of MMPs in biology and of the previous efforts to target MMPs as therapeutics (Chapter 1), (b) describe our efforts at over-expression, purification, and characterization of the catalytic domains of MMP7 (cdMMP7) and MMP16 (cdMMP16) (Chapters 2 and 3), (c) present our efforts at the preparation and initial spectroscopic characterization of Co(II)-substituted analogs of cdMMP7 and cdMMP16 (Chapters 2 and 3), (d) present inhibition data on cdMMP7 and cdMMP16 using zinc binding groups (ZBG) as potential scaffolds for future inhibitors (Chapter 3), and (e) summarize our data in the context of previous results and suggest future directions (Chapter 4). The work described in this dissertation integrates biochemical (kinetic assays, inhibition studies, limited computational methods), spectroscopic (CD, UV-Vis, 1H-NMR, fluorescence, and EXAFS), and analytical (MALDI-TOF mass spectrometry, isothermal calorimetry) methods to provide a detailed structural and mechanistic view of these MMPs.
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function, and “Omic
    Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ †Life Sciences Institute, Department of Oral Biological and Medical Sciences, and ‡Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms of
    [Show full text]
  • Topfind 2.0—Linking Protein Termini with Proteolytic Processing and Modifications Altering Protein Function Philipp F
    Published online 18 November 2011 Nucleic Acids Research, 2012, Vol. 40, Database issue D351–D361 doi:10.1093/nar/gkr1025 TopFIND 2.0—linking protein termini with proteolytic processing and modifications altering protein function Philipp F. Lange1,2,3,*, Pitter F. Huesgen1,2,3 and Christopher M. Overall1,2,3,* 1Centre for Blood Research, 2Department of Oral Biological and Medical Sciences and 3Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 Received August 26, 2011; Revised October 6, 2011; Accepted October 23, 2011 ABSTRACT INTRODUCTION The function and morphology of a cell is by and large Protein termini provide critical insights into the Downloaded from functional state of individual proteins. With recent carried out by its proteome, likewise, that of a tissue by advances in specific proteomics approaches to its cells and their proteomes. The functional state of the enrich for N- and C-terminomes, the global proteome, and hence a cell, is defined by the structures, interactions and post-translational modifications of the analysis of whole terminomes at a proteome-wide proteins present at any given time and place. Proteins scale is now possible. Information on the actual N- are synthesized as single polypeptide chains that have a http://nar.oxfordjournals.org/ and C-termini of proteins in vivo and any start represented by the amino (N-) terminus, and a post-translational modifications, including their carboxyl (C-) terminus forming its end. Protein termini generation by proteolytic
    [Show full text]
  • CLIPPER: an Add-On to the Trans-Proteomic Pipeline for the Automated Analysis of TAILS N-Terminomics Data
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library DOI 10.1515/hsz-2012-0269 Biol. Chem., 2012; 393(12): 1477–1483 Short Communication Ulrich auf dem Keller * and Christopher M. Overall CLIPPER: an add-on to the Trans-Proteomic Pipeline for the automated analysis of TAILS N-terminomics data Abstract: Data analysis in proteomics is complex and with conditions, which has been hampered until recently by the extra challenges involved in the interpretation of data the lack of appropriate technologies (Overall and Blobel , from N-terminomics experiments, this can be daunting. 2007 ). To address this limitation several novel proteom- Therefore, we have devised a rational pipeline of steps to ics methods for the system-wide discovery of protease approach N-terminomics data analysis in a statistically- substrates in complex biological samples have been based and valid manner. We have automated these steps introduced (auf dem Keller and Schilling , 2010 ; Impens in CLIPPER, an add-on to the Trans-Proteomic Pipeline et al. , 2010 ; Huesgen and Overall , 2012 ) in the subfield of (TPP). Applying CLIPPER to the analysis of N- terminomics proteomics that has come to be known as degradomics data generated by terminal amine isotopic labeling of (Lopez -Otin and Overall, 2002 ). substrates (TAILS) enables high confidence peptide to High throughput degradomics techniques aim for protein assignment, protein N-terminal characterization the enrichment and, ideally, relative quantification of and annotation, and for protease analysis readily allows N-terminal peptides in protease-treated and control protease substrate discovery with high confidence.
    [Show full text]
  • The Podocyte Protease Web: Uncovering the Gatekeepers of Glomerular Disease
    The Podocyte Protease Web: Uncovering the Gatekeepers of Glomerular Disease Markus M. Rinschen 1,2,3, Pitter F. Huesgen 4, Rachelle E. Koch 5 1 Department II of Internal Medicine, and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 2 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. 3 Center for Mass Spectrometry and Metabolomics, The Scripps Research Institute, La Jolla, CA, USA 4 Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany 5 Division of Graduate Medical Sciences, Boston University School of Medicine, Boston, MA Address correspondence to: [email protected], [email protected] Joseph-Stelzmann-Str. 26 50931 Cologne Germany Fax +49 221/478 1426623 This manuscript was accepted published online by the American Journal of Physiology- Renal Physiology on Sept. 19, 2018. The final version of the manuscript is available at: doi.org/10.1152/ajprenal.00380.2018 Abstract Proteases regulate glomerular physiology. The last decade has revealed a multitude of podocyte proteases that govern the glomerular response to numerous physical and biological cues. These proteases form a protein signaling web that generates stress stimuli and serves as a key controller of the glomerular microenvironment. Furthermore, both the extracellular and intracellular proteolytic networks are perturbed in focal segmental glomerulosclerosis, as well as hypertensive and diabetic nephropathy. The highly-intertwined podocyte protease web is accordingly an integrative part of the podocyte’s damage response and is reactive to a variety of chemical, mechanical, and metabolic cues. Novel mass-spectrometry based technologies will help to untangle this proteolytic network: functional readouts acquired from deep podocyte proteomics, single glomerular proteomics, and degradomics may expose unprecedented protease activity.
    [Show full text]
  • Comprehensive Analysis of Human Protein N-Termini Enables
    www.nature.com/scientificreports OPEN Comprehensive analysis of human protein N-termini enables assessment of various protein Received: 28 February 2017 Accepted: 9 June 2017 forms Published online: 26 July 2017 Jeonghun Yeom1,2, Shinyeong Ju1,3, YunJin Choi4, Eunok Paek 4 & Cheolju Lee1,2 Various forms of protein (proteoforms) are generated by genetic variations, alternative splicing, alternative translation initiation, co- or post-translational modifcation and proteolysis. Diferent proteoforms are in part discovered by characterizing their N-terminal sequences. Here, we introduce an N-terminal-peptide-enrichment method, Nrich. Filter-aided negative selection formed the basis for the use of two N-blocking reagents and two endoproteases in this method. We identifed 6,525 acetylated (or partially acetylated) and 6,570 free protein N-termini arising from 5,727 proteins in HEK293T human cells. The protein N-termini included translation initiation sites annotated in the UniProtKB database, putative alternative translational initiation sites, and N-terminal sites exposed after signal/transit/pro- peptide removal or unknown processing, revealing various proteoforms in cells. In addition, 46 novel protein N-termini were identifed in 5′ untranslated region (UTR) sequence with pseudo start codons. Our data showing the observation of N-terminal sequences of mature proteins constitutes a useful resource that may provide information for a better understanding of various proteoforms in cells. Te diversity of protein from a single gene is generated by several mechanisms, including co- or post-translational modifcation, alternative splicing and alternative translation initiation. Tese processes afect function, localiza- tion, or stability of the protein. In particular, there may be a signifcant disparity between N-terminal site pre- dicted at the gene level and the N-terminal status of the protein product.
    [Show full text]
  • UC San Francisco Electronic Theses and Dissertations
    UCSF UC San Francisco Electronic Theses and Dissertations Title Identification and exploration of apoptotic and caspase proteolytic substrates. Permalink https://escholarship.org/uc/item/86t0d35v Author Seaman, Julia Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California ii Dedication To my husband, Nate, and my family, Mom, Dad and Chris. iii Acknowledgements I am very grateful for the advice, assistance and support from the Wells Lab. My advisor, Jim Wells, has created a wonderful laboratory environment for collaborative research and friendships. The lab, and members in it, could not have survived without the innumerable skills of Marja Tarr. My graduate career has been greatly assisted by sharing the experiences with fellow students Ashley Smart and Charlie Morgan in the lab. It has been fun to explore science and solve the big and small problems with them and the other graduate students in lab- Justin Rettenmaier, Hai Tran, Sam Pollock, and Alex Martinko. I have relied heavily on everyone else who counts himself or herself as a Wellsome member. The postdocs- especially Kazu Shimbo, Nick Agard, Arun Wiita, Jason Porter, Juan Diaz, JT Koerber, Nathan Thomsen, Duy Ngyuen, Peter Lee, Min Zhuang, Zach Hill and Olivier Julien- have helped improved my scientific skills and graduate school experience throughout my time in the lab. Finally, my scientific work relies heavily on many members who came before me. UCSF has been a great environment to learn and explore during my PhD. I am very thankful my program, PSPG and its staff- Deana Kroetz, Debbie Acoba and Rebecca Brown.
    [Show full text]
  • Terminomics Methodologies and the Completeness of Reductive Dimethylation: a Meta-Analysis of Publicly Available Datasets
    proteomes Technical Note Terminomics Methodologies and the Completeness of Reductive Dimethylation: A Meta-Analysis of Publicly Available Datasets Mariella Hurtado Silva 1 , Iain J. Berry 1,2, Natalie Strange 1 , Steven P. Djordjevic 2 and Matthew P. Padula 1,* 1 Proteomics Core Facility and School of Life Sciences, Faculty of Science, University of Technology Sydney, Broadway NSW 2007, Australia; [email protected] (M.H.S.); [email protected] (I.J.B.); [email protected] (N.S.) 2 The ithree Institute, Faculty of Science, University of Technology Sydney, Broadway NSW 2007, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-403-838-981 Received: 16 January 2019; Accepted: 25 March 2019; Published: 29 March 2019 Abstract: Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the labelling was not complete and investigated whether this was a common occurrence. We assessed the completeness of labelling in a selection of existing, publicly available N-terminomics datasets and empirically determined that amine-based labelling chemistry does not achieve complete labelling and potentially has issues with labelling amine groups at sequence-specific residues. This finding led us to conduct a thorough review of the historical literature that showed that this is not an unexpected finding, with numerous publications reporting incomplete labelling. These findings have implications for the quantitation of N-terminal peptides and the biological interpretations of these data.
    [Show full text]