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Dissertation Kollipara.Pdf Characterizing protein processing in the Endoplasmic Reticulum using quantitative proteomics: the pathogenesis of the Marinesco-Sjörgren Syndrome Zur Erlangung des akademischen Grades eines Dr. rer. nat von der Fakultät Bio- und Chemieingenieurwesen der Technischen Universität Dortmund genehmigte Dissertation vorgelegt von MSc. Laxmikanth Kollipara aus Hyderabad, India Tag der mündlichen Prüfung: 20.12.2016 1. Gutachter: Prof. Dr. Albert Sickmann 2. Gutachter: Prof. Dr. Oliver Kayser Dortmund 2016 1. Prüfer: Prof. Dr. Markus Nett Abstract Abstract In this work, characterization of Marinesco-Sjögren Syndrome (MSS) was performed for the first time on the proteome level using mass spectrometry (MS)-based quantitative proteomics strategies. MSS is a neuromuscular and neurodegenerative disorder and it is caused due to the mutational inactivation of SIL1 protein, which results in malfunctioning of protein folding machinery mediated by the chaperone BiP that can lead to the ER stress-induced cell death via apoptotic signaling. The major goals were (i) to understand the rescue mechanisms in SIL1-deficient non-vulnerable tissues from human and (ii) to verify the cellular perturbations caused due to the loss of functional SIL1 in woozy mouse (i.e. mouse model of MSS). To achieve these aims, samples derived from five different MSS cases and two different tissues from woozy along with their respective healthy controls were studied. For this, comparative LC-MS proteomics approaches such as chemical labeling (i.e. iTRAQ) and label- free quantification (precursor ion intensity based and NSAF) were employed. During which, sample preparation workflows were optimized that enabled to process clinical samples related to MSS that included primary cell lines and mammalian tissues for the subsequent quantitative LC-MS analyses. This also included an investigation of occurrence of artificial protein carbamylation, which is a well- known unwanted artefact in quantitative proteomics. By employing these workflows, abundances of thousands of proteins in both MSS patients and woozy were relatively quantified. Among these, the number of differentially regulated proteins varied depending on the cell/tissue type and the clinical state i.e. SIL1-affected or unaffected. However, in both conditions, the absence of functional SIL1 showed disturbed cellular activities suggesting its important role in BiP-mediated protein folding process. In MSS SIL1 non-vulnerable tissues data, the processes which might mitigate the ER-stress induced due to SIL1 loss were identified. Next, proteome analysis of SIL1 depleted HEK293 cell line was performed to study the pathophysiology of SIL1 loss in more detail. Additionally, a targeted-MS based method was developed to assay proteins that are involved in the unfolded protein response pathway, which is triggered under the ER-stress conditions. Lastly, proteomic profiling of a human myoblastic RCMH cell line was carried out that can serve as an in vitro model to investigate muscle and neuromuscular disorders. Abstract (in German) Abstract (in German) In dieser Arbeit wurde eine Charakterisierung des Marinesco-Sjörgen Syndroms (MSS) zum ersten Mal auf Proteom-Ebene mittels Massenspektrometrie-basierten, quantitativen Methoden durchgeführt. MSS ist eine neuromuskuläre und neurodegenerative Erkrankung und wird verursacht durch mutations-bedingte Inaktivierung des SIL1 Proteins. Diese führt zu einer Störung der Proteinfaltung durch das Chaperonprotein BiP und somit zu einer ER-Stresssituation mit möglichem folgendem Zelltod durch Apoptose. Das Hauptziel bestand darin, die Rettungsmechanismen in SIL1 unabhängigen Geweben (human) zu verstehen, sowie bei fehlendem funktionellen SIL1 in woozy Mäusen (MSS-Mausmodell) die verursachten Störungen auf zellulärer Ebene zu bestimmen. Hierzu wurden vergleichende, proteomische Strategien, wie chemische Isotopenmarkierung und Label-freie Quantifizierung angewandt. Innerhalb der Durchführung wurden Methoden der Probenvorbereitung weiter optimiert, um eine effektive Aufbereitung von klinischen Proben im Zusammenhang mit MSS und auch von Primärzelllinien sowie Säugerzellgeweben für anschließende, quantitative LC-MS Analytik zu ermöglichen. Durch Nutzung dieser erstellten Arbeitsanweisungen, konnten tausende von Proteinen in MSS- Patienten und woozy relativ quantifiziert werden. Unter diesen Proteinen variierte die Menge von differenziell regulierten Proteinen abhängig von Zell- und Gewebstyp und vom klinischen Zustand, insbesondere durch deren Abhängigkeit von SIL1 oder Unabhängigkeit davon. Dennoch zeigte sich bei Abwesenheit von funktionellem SIL1 eine Störung der zellulären Aktivität in beiden Zuständen (SIL1 abhängig oder unabhängig). Dies deutet auf eine wichtige Funktion von SIL1 in der BiP- vermittelten Proteinfaltung hin. In MSS SIL1 unabhängigem Gewebe wurden die Prozesse, welche womöglich die Reduktion des ER-Stresszustandes herbeiführen und durch SIL1-Verlust induziert ist, identifiziert. Anschließend wurde eine Proteomanalyse von SIL1 knock-down HEK293 Zelllinien durchgeführt, um die Pathophysiologie im Falle von SIL1-Verlust im Detail zu untersuchen. Weiterhin wurde eine targeted-MS basierte Strategie entwickelt, um Proteine zu studieren, welche involviert sind in Signalwegen der Antwort auf ungefaltete Proteine und im Fall von ER- Stresssituation hervorgerufen wird. Letztlich wurde eine proteomische Analyse einer humanen RCMH Zelllinie durchgeführt. Diese Zelllinie kann womöglich als ein in vitro Modell zur Untersuchung von muskulären und neuromuskulären Fehlsteuerungen dienen. Table of contents Table of contents Abstract ............................................................................................................................ 3 Abstract (in German) ........................................................................................................ 4 Abbreviations ................................................................................................................... 8 Chemical structures of amino acids ................................................................................... 9 List of publications .......................................................................................................... 10 List of poster presentations ............................................................................................. 12 1 Introduction ............................................................................................................. 13 1.1 Endoplasmic reticulum and protein folding process ....................................... 13 1.1.1 SIL1-BiP chaperone system .............................................................................. 14 1.2 ER stress, homeostasis and apoptosis ............................................................. 16 1.3 Protein aggregation related mammalian disorders ........................................ 18 1.3.1 Marinesco-Sjögren Syndrome ......................................................................... 19 1.3.2 Animal model of MSS ...................................................................................... 23 1.4 Proteome analysis - in general ........................................................................ 24 1.4.1 Mass spectrometry - based proteomics .......................................................... 25 1.4.1.1 Electrospray ionization .............................................................................. 26 1.4.1.2 Reversed-phase chromatography .............................................................. 27 1.4.1.3 Off-line fractionation strategies ................................................................. 28 1.4.1.4 Mass analyzers ........................................................................................... 30 1.4.1.5 Tandem mass spectrometry ...................................................................... 34 1.4.1.6 MS data acquisition strategies ................................................................... 36 1.4.1.7 Database dependent protein identification .............................................. 36 1.4.2 Quantitative proteomics to study human diseases ......................................... 38 1.4.2.1 Protein quantification with labeling reagents ........................................... 39 1.4.2.2 Label-free protein quantification ............................................................... 42 1.4.2.3 Protein quantification with targeted MS ................................................... 44 2 Aim .......................................................................................................................... 46 3 Materials and methods ............................................................................................ 47 3.1 Materials .......................................................................................................... 47 3.1.1 Chemicals ......................................................................................................... 47 3.1.2 Instruments and disposable consumables ...................................................... 48 3.1.3 LC-columns, HPLCs and mass spectrometers .................................................. 48 3.1.4 Data analysis software ..................................................................................... 49 3.2 Methods ........................................................................................................... 49 3.2.1 Cell and tissue samples for proteomics analyses ...........................................
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