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Proteome-Wide Identification and Characterization of Protein ADP-Ribosylation in Mammalian Cells and Mouse Tissues

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Proteome-Wide Identification and Characterization of Protein ADP-Ribosylation in Mammalian Cells and Mouse Tissues Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 Proteome-wide identification and characterization of protein ADP-ribosylation in mammalian cells and mouse tissues Leutert, Mario Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-142856 Dissertation Published Version Originally published at: Leutert, Mario. Proteome-wide identification and characterization of protein ADP-ribosylation inmam- malian cells and mouse tissues. 2017, University of Zurich, Faculty of Science. Proteome-wide Identification and Characterization of Protein ADP-ribosylation in Mammalian Cells and Mouse Tissues Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Mario Peter Leutert von Schaffhausen, SH Promotionskommission Prof. Dr. Dr. Michael O. Hottiger (Vorsitz und Leitung der Dissertation) Prof. Dr. Alessandro A. Sartori Prof. Dr. Bernd Wollscheid Prof. Dr. Alexander Bürkle Zürich, 2018 SUMMARY In all organisms the functional and structural plasticity of proteins is defined by post-translational modifications (PTM). ADP-ribosylation is an ancient PTM that comes in a monomeric and polymeric form in mammalian cells and has been identified to be involved in important cellular processes and misregulated in disease. Inhibitors of ADP-ribosylation are now used in the clinic to treat cancer, however many aspects of the PTM remain enigmatic; especially which proteins are modified during which conditions, what are the amino acid acceptor sites and which sites are modified by which ADP-ribosyltransferase. The aim of this thesis was to develop and apply mass spectrometry based methods to systematically and accurately map ADP-ribosylation sites in cells and tissues during physiological and pathological conditions and characterize their biological function. First, we established a new enrichment protocol and optimized the mass spectrometry method, which let to the identification of the oxidative stress-induced ADP-ribosylome in HeLa cells and the finding that serine serves as the major nuclear ADP-ribose acceptor site. The modified proteins were mostly associated with functions in regulation of transcription, chromosome organization as well as DNA and RNA metabolic processes. We also found that a small number of tyrosines are ADP-ribosylated. Furthermore we identified high levels of arginine ADP-ribosylated proteins in mouse liver extracts on proteins having a role in metabolism. Second, we found that ARTD1 is the main mediator of nuclear ADP- ribosylation during oxidative stress in cultured cells, having selectivity for serine and tyrosine modification. ARTD2 only modified a small number of detected targets. This suggested that ARTD1 catalyzes poly-ADP-ribosylation as well as de novo protein ADP-ribosylation. Third we mapped the ADP-ribosylome of heart and skeletal muscle tissue and revealed a widespread role of ARTC1 arginine ADP-ribosylation of extracellular and plasma membrane proteins, as well as ARTC1 independent ADP-ribosylation in mitochondria. Together, we could identify enzyme-substrate relationships for several hundred ADP-ribosylation sites catalyzed by ARTD1, ARTD2, ARTC1 and ARTC2.2 and we got mechanistic insight into the function of a few selected ADP-ribosylation sites. We are now able to accurately localize ADP-ribosylation sites and to map tissue and treatment specific ADP-ribosylation sites. 1 ZUSAMMENFASSUNG Funktionelle und strukturelle Proteineigenschaften können in allen Organismen durch post-translationale Modifikationen (PTM) beeinflusst werden. ADP- Ribosylierung ist eine PTM, welche sowohl als Mono- als auch als Polymer vorkommt, in wichtige zelluläre Prozesse involviert ist und unter pathologischen Konditionen fehlreguliert sein kann. Obwohl Inhibitoren von ADP-Ribosylierung in der Krebsbehandlung eigesetzt werden, bleiben viele Aspekte unklar. Es ist ungenügend geklärt, welche Proteine unter welchen Bedingungen modifiziert sind, welche Aminosäuren als Akzeptoren fungieren und inwieweit die verschiedenen ADP-Ribosyltransferasen in die Etablierung der Modifikation involviert sind. Das Ziel dieser Doktorarbeit war es, massenspektrometrische Methoden zu entwickeln, um systematisch und präzise ADP-Ribosylierungsstellen im Proteom von Zellen und Geweben zu kartieren und deren biologische Funktion zu analysieren. In einem ersten Schritt haben wir ein neues Anreicherungsprotokoll für ADP- ribosylierte Peptide etabliert und die massenspektrometrische Methode dafür optimiert. Mithilfe dieses Protokolls, war es uns gelungen, das durch oxidativen Stress induzierte ADP-Ribosylom in HeLa Zellen zu identifizieren. Die modifizierten Proteine spielen eine Rolle in der transkriptionellen Regulation, Chromosomen Organisation sowie in DNA und RNA metabolischen Prozessen. In einem zweiten Schritt konnten wir zeigen, dass ARTD1 der Hauptregulator der von oxidativem Stress induzierten, nukleären ADP-Ribosylierung war und Proteine selektiv an Serin oder Tyrosin modifizierte. ARTD2 war dagegen nur für die Modifikation einer kleinen Anzahl von unterschiedlichen identifizierten Proteinen verantwortlich. In einem dritten Schritt haben wir das ADP-Ribosylom von Herz- und Skelettmuskeln der Maus charakterisiert. Wir konnten zeigen, dass ARTC1 eine weitreichende Rolle in der Arginin-spezifischen ADP-Ribosylierung von Membranproteinen, sowie extrazellulären Proteinen spielte. Im Gegensatz dazu, haben wir auch eine ARTC1 unabhängige, mitochondriale ADP-Ribosylierung beobachtet. Diese Daten ermöglichten es uns, Substrat-Enzym-Beziehungen für mehrere hundert ARTD1-, ARTD2-, ARTC1- und ARTC2.2-abhängige ADP- Ribosylierungsstellen zu erstellen und die funktionelle Relevanz der Modifikation für einige Proteine zu studieren. Wir sind nun in der Lage ADP-ribosylierte Proteine und deren Modifikationsstellen präzise zu identifizieren und gewebe- und behandlungsspezifische ADP-Ribosylierungsstellen zu charakterisieren. 2 TABLE OF CONTENTS SUMMARY ................................................................................................................................................. 1 ZUSAMMENFASSUNG ............................................................................................................................. 2 TABLE OF CONTENTS ............................................................................................................................ 3 INTRODUCTION ...................................................................................................................................... 6 1 PROTEOFORMS AND PROTEOME COMPLEXITY ............................................................................................. 6 2 PROTEIN ADP-RIBOSYLATION ........................................................................................................................ 8 3 ADP-RIBOSYLTRANSFERASES ......................................................................................................................... 8 3.1 Prokaryotic ADP-ribosylating toxins ............................................................................................... 8 3.2 ARTD family of eukaryotic ADP-ribosyltransferases .............................................................. 10 3.3 ARTC family of eukaryotic ADP-ribosyltransferases ............................................................... 11 3.4 Sirtuins ........................................................................................................................................................ 12 3.5 NAD+ metabolism and ADP-ribosyltransferases ...................................................................... 12 4 ADP-RIBOSYLHYDROLASES .......................................................................................................................... 13 5 ADP-RIBOSYLATION INTERACTORS ........................................................................................................... 14 6 BIOLOGICAL ROLES OF INTRACELLULAR ADP-RIBOSYLATION .............................................................. 15 6.1 Biological functions of PARylation ................................................................................................. 15 6.2 PARP inhibitors as therapeutics ...................................................................................................... 17 7 FUNCTIONAL ROLES OF EXTRACELLULAR ADP-RIBOSYLATION ............................................................ 18 8 LARGE-SCALE ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS BY MASS SPECTROMETRY ................................................................................................................................................. 20 8.1 Identification of PTMs by mass spectrometry ............................................................................ 20 8.2 Enrichment of PTM carrying peptides .......................................................................................... 22 8.3 Interpreting PTMs by proteomics exemplified by phosphoproteomics .......................... 23 9 PROTEOME-WIDE IDENTIFICATION OF ADP-RIBOSYLATION BY MASS SPECTROMETRY .................. 24 9.1 Challenges in identifying the ADP-ribosylome .......................................................................... 24 9.2 Affinity purification of ADP-ribosylated proteins .................................................................... 25
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