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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Für Proteomik Und Bioanalytik

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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Für Proteomik Und Bioanalytik TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Proteomik und Bioanalytik Regulation of Protein Translation and Degradation in Neurons Ruchi Chauhan Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. M. Klingenspor Prüfer der Dissertation: 1. Univ.-Prof. Dr. B. Küster 2. Assist. Prof. Dr. J. Steen Die Dissertation wurde am 02.02.2015 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 21.05.2015 angenommen. i To those who believed in me and demanded perfection (Gayatri, Ved, Judith, Late V.K. Jain and Soham) ii Table of Contents Contents Page No. Abstract iv-v Zusammenfassung vi-vii Chapter 1: Introduction to Protein Homeostasis in Neurons 1-42 Chapter 2: Quantitative Profiling of Peptides from RNAs 43-93 classified as noncoding Chapter 3: Co-regulation Proteomics Reveals Substrates 94-116 and Mechanisms of APC/C complex dependent degradation Chapter 4: The APC/C complex Regulates HuD protein interaction 117 -139 with SMN protein in SMA disease Chapter 5: The APC/C complex Regulates SMN protein and its inhibition 140- 168 Rescues SMA disease Chapter 6: General Discussion and Future Perspectives 169-177 List of Publications 179 Acknowledgements 180 Curriculum vitae 181-182 Lebenslauf 183-184 iii Abstract Regulation of cellular proteome by protein translation and degradation, protein homeostasis, is crucial for the normal functioning of a cell - especially, in a highly metabolic and active cell such as the neuron. Perturbations in protein homeostasis have been implicated in various neuronal diseases. Abnormal regulation of even a single protein e.g. Survival of Motor Neurons (SMN) may cause debilitating neurodegenerative disease such as Spinal Muscular Atrophy (SMA). In this thesis, I undertook global high-throughput quantitative mass-spectrometry based proteomics studies, along with biochemical validation of subset of proteins, to understand protein homeostasis in neurons. The specific aim of this study was to investigate (a) activity dependent protein synthesis in stimulated neurons, (b) global protein degradation by Anaphase Promoting Complex/Cyclosome (APC/C) mediated ubiquitination mechanisms and (c) detailed regulation of SMN protein along with its interacting partner HuD by APC/C in the context of SMA disease. The results from these studies provide novel insights in understanding the regulation of protein translation and degradation in neurons, and may also have potential for developing therapeutics. ‘Novel’ peptides/translational products were identified from non-coding regions of the genome in depolarized cortical neurons from mouse. The abundance of these peptides was regulated over time similar to that of known proteins, indicating that ‘novel’ proteome in these neurons may have biological functions. In the protein degradation study, new members of the kinesin family were observed to be undergoing APC/C mediated degradation. The degradation of KIFC1, a kinesin protein, by APC/C was regulated by phosphorylation event. In the final systematic study to identify molecular mechanisms that may regulate SMN protein stability, its novel interaction with the E3 ligase APC/C in neurons was observed using proteomics. Also, we confirmed the earlier known interaction of HuD protein with SMN and provided new evidence that down- regulation of HuD protein concurs with low levels of SMN protein in neurons indicating that HuD may be stabilizing SMN protein. Interestingly, overexpression of HuD in the zebrafish model of SMA partially rescues the disease phenotype, thus suggesting the direct involvement of HuD protein in the SMA. Further, novel interaction of HuD with APC/C was observed using proteomics and SMN and HuD proteins were observed to be undergoing ubiquitination and degradation by APC/C. However, the mechanisms by which APC/C ubiquitinates these proteins were identified to be different. SMN degradation, in vitro, was regulated by APC/C-mediated ubiquitination, which in turn depends on phosphorylation of SMN protein. However, HuD iv ubiquitination and degradation, in vitro, was regulated by the presence of a D-box motif in its sequence. We then employed multi-pronged approaches for inhibition of APC, including a small molecule inhibitor, proTAME, which increase the SMN protein abundance as well as that of HuD protein in mouse neurons. More importantly, the APC/C inhibition in the zebrafish model of SMA was shown to rescue the disease phenotype remarkably, along with a detectable increase in HuD and SMN protein levels. Thus, results from these studies have far-reaching implications for future studies on protein homeostasis in neurons and especially protein synthesis by non- canonical translational events. I also discussed the importance of identifying novel substrates of the APC/C in cancer such as KIFC1 as well as neuronal disorders such as HuD and SMN along with molecular mechanisms crucial in understanding the SMA disease and finding the potential cure. v Zusammenfassung Proteinhomöostase ist die Regulierung des zellulären Proteoms durch Translation und durch Abbau von Proteinen und ist entscheidend für die normale Funktion der Zelle vorallem in hoch metabolischen und aktiven Zellen wie den Nervenzellen. Störungen der Proteinhomöostase wurden bererits mit verschiedensten neuronalen Erkrankungen in Verbindung gebracht. Abnormale Regulierungen von nur einem Protein, z.B. Survival of Motor Neurons (SMN), können neurodegenerative Krankheiten der spinalen Muskelatrophie (SMA) verursachen. In dieser Arbeit wurde eine auf quantitativer Massenspektrometrie basierenden high-troughput Methoden zur Untersuchung des Proteoms zusammen mit einer detailierten biochemischen Validierung ausgewählter Proteine durchgeführt um die Proteinhomöostase in Nervenzellen zu untersuchen. Das Ziel dieser Arbeit a) die aktivitätsabhängige Proteinsynthese in stimulierten Nervenzellen, b) den globalen Proteinabbau durch den Anaphase Promoting Complex/Cyclosome (APC/C) mit Hilfe von Ubiquitinierung und c) die detaillierte Regulierung des SMN Proteins gemeinsam mit dessen interagierenden Partner HuD durch APC/C im Zusammenhang mit SMA Erkrankungen zu untersuchen. Die erhaltenen Resultate zeigen einen neuen Einblick und führen zu einem besseren Verständnis der Regulierung von Translation und Abbau von Proteinen in Nervenzellen. Des Weiteren können diese Ergebnisse die Entwicklung neuer Therapeutika ermöglichen. Peptide bzw. translatorische Produkte von nicht kodierenden Regionen des Genoms in depolarisierten, kortikalen Nervenzellen der Maus wurden identifiziert. Die Menge an Peptiden wurde über die Zeit ähnlich reguliert wie bei bekannten Proteinen und dies deutet auf die Anwesenheit eines „neuen“ Proteoms hin welches biologische Funktionen besitzt kann. Mit Hilfe der Untersuchung des Proteinabbaus wurden neue Mitglieder der Kinesin Proteine, welche unerstuetzt durch APC/C abgebaut werden, identifiziert und validiert. Weiters wurde dadurch auch gezeigt, dass der Abbau des KIFC1 Proteins mittels APC/C abhängig ist von Phosphorilierungsereignissen. Während der gezielten Untersuchung des Proteoms zur Identifizierung des molekularen Mechanismus, welcher möglicherweise die Stabilität des SMN Protein reguliert, wurde eine neue Interaktion mit der E3 Ligase APC/C in Nervenzellen festgestellt. Die bereits bekannten Interaktionen des HuD Proteins mit SMN wurden ebenfalls bestätigt. Die erhaltenen Ergebnisse zeigen, dass die verminderte Expression des HuD Proteins mit einem geringen Level des SMN Proteins in Nervenzellen einhergeht, welches darauf hinweist dass HuD das SMN Protein stabilisiert. Die Überexpression von HuD im vi Zebrafischmodell mit SMA verbessert teilweise den Verlauf des Krankheitsphenotyp und dieses unterstützt die Rolle des HuD Protein in SMA. Weiters wurden mit Hilfe von Proteomics neue Interaktionen von HuD mit APC gezeigt und es wurde entdeckt, dass SMN und HuD Proteine von APC/C ubiquitiniert und abgebaut werden. Jedoch zeigte sich, dass der Mechanismus mit welchem APC die Proteine ubiquitiniert unterschiedlich ist und das der Abbau von SMN in vitro durch Ubiquitinierung reguliert ist welche wiederum abhängig ist von der Phosphorylierung des SMN Proteins. Im Gegensatz dazu is die Ubiquitinierung von HuD und deren Abbau reguliert durch das Vorhandensein eines D-Box Motives in deren Sequenz. Anschliessend wurden mehrstufige Methoden zur Inhibierung von APC angewendet welche unter anderem den Inhibitor proTAME beinhalten. Dieser Inhibitor erhöht die Menge an SMN und HuD Protein in den Nervenzellen der Maus. Von grosser Bedeutung ist auch, dass gezeigt wurde, dass durch die Inhibierung von APC im Zebrafisch Modell mit SMA der Krankheitshenotyp gerettet werden kann einhergehend mit dem detektierbaren Erhöhung des HuD und SMN Proteinlevels. Diese Arbeit ermöglicht somit weitreichende Anwendungen für zukünftige Untersuchungen von Proteinhomöostase in Nervenzellen und im speziellen fuer die Proteinsynthese durch atypische Translationsereignisse. In dieser Arbeit wurde auch die Wichtigkeit der Identifikation von neuen APC/C Substraten in Krebs und neuronalen Störungen diskutiert sowie molekulare Mechanismen welche notwendig sind um SMA Erkrankungen zu untersuchen und potentielle Heilmittel zu finden. vii Chapter 1 Introduction
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