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Studie Oxidativer Veränderungen Während Der Neuronalen Differenzierung Von SH-SY5Y-Zellen Mithilfe Der Proteomanalyse

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Studie Oxidativer Veränderungen Während Der Neuronalen Differenzierung Von SH-SY5Y-Zellen Mithilfe Der Proteomanalyse Studie oxidativer Veränderungen während der neuronalen Differenzierung von SH‐SY5Y‐Zellen mithilfe der Proteomanalyse Inaugural‐Dissertation zur Erlangung des Doktorgrades der Mathematisch‐Naturwissenschaftlichen Fakultät der Heinrich‐Heine‐Universität Düsseldorf vorgelegt von Katrin Charlotte Brenig aus Linnich Düsseldorf, November 2018 aus dem Molecular Proteomics Laboratory der Heinrich‐Heine‐Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch‐Naturwissenschaftlichen Fakultät der Heinrich‐Heine‐Universität Düsseldorf Berichterstatter: 1. Prof. Dr. K. Stühler 2. Prof. Dr. D. Willbold Tag der mündlichen Prüfung: 02. Mai 2019 Mathilda & Julian Zusammenfassung I Zusammenfassung Ein gängiges Modellsystem um initiale Prozesse der neuronalen Differenzierung zu untersuchen, ist die humane Neuroblastom‐Zelllinie SH‐SY5Y. Obwohl die durch Retinsäure induzierten Vorgänge zunehmend mit redox‐sensitiven Prozessen in Verbindung gebracht werden, sind die molekularen Auswirkungen auf die Proteine nicht vollständig aufgeklärt. In dieser Arbeit wurde daher der Einfluss von oxidativen Veränderungen während der frühen Zeitpunkte der neuronalen Differenzierung des Zellmodells mithilfe der Proteomanalyse untersucht. Infolge des angewandten Differenzierungsprotokolls kommt es zu einer signifikanten Extension der Neuriten, einem phänotypischen Merkmal des neuronalen Differenzierungsprozesses. Mittels quantitativer Massenspektrometrie wurden zwei Proteomanalysen durchgeführt, welche die differenziellen Proteinabundanzen in einem dynamischen Verlauf zwischen 0 ‐ 48 Std. und nach 120 Std. darstellen. Dadurch konnten veränderte Abundanzen von Markerproteinen nachgewiesen werden, die am Retinsäure‐Signalweg (z.B. CRABP2) und der neuronalen Differenzierung (z.B. RET) beteiligt sind. Unterstützt wurden diese Ergebnisse durch quantitative mRNA Daten. Einhergehend mit der größten morphologischen Veränderung nach 48 Std. legen auch die Proteomanalysen nahe, dass die Zellen von einem proliferierenden in einen differenzierenden Zustand übergehen. Die, mithilfe von redox‐sensitiven Sensoren (RoGFP2), durchgeführten Experimente weisen schließlich auf einen signifikanten Anstieg von zytosolischen H2O2 nach zweistündiger Retinsäurebehandlung hin, an dem wahrscheinlich eine Aktivität des NADPH‐Oxidase‐Komplexes‐2 beteiligt ist. Für die globale Analyse von reversibel oxidierten Proteinen wurde ein redox‐spezifisches Proteinanreicherungs‐ protokoll etabliert, welches einen Nachweis mittels quantitativer Massenspektrometrie ermöglichte. Während zwischen 0 ‐ 3 Std. nur < 0,5% der Proteine eine Veränderung in der Abundanz aufwiesen, zeigten im Redox‐Proteom nach 2 Std. Retinsäurebehandlung 275 (16%) der 1 769 quantifizierten cysteinhaltigen Proteine signifikante Unterschiede. Darunter solche mit bereits bekanntem Bezug zu redox‐sensitiven Prozessen und/oder der neurologischen Entwicklung (z. B. RAC1). Für 57 dieser 275 Proteine lieferten die Ergebnisse zudem Informationen über die potenziell reversibel oxidierten Cysteine. Schließlich wurde gezeigt, dass eine Veränderung des zellulären Redoxzustandes, durch Zugabe des Reduktionsmittels N‐Acetylcystein oder des Radikalfängers Butylhydroxyanisol, sowohl Auswirkungen auf die Abundanz von Markerproteinen hat als auch zu signifikant verkürzten Neuriten führt. Dieses Experiment ist ein weiterer Hinweis darauf, dass zelluläre Redoxprozesse an der neuronalen Differenzierung des Zellmodells beteiligt sind. Im Rahmen dieser Arbeit wurden zwei Modelle vorgeschlagen, die die molekularen Auswirkungen veränderter ROS‐Mengen erklären. (1) ROS sind Teil des Autoregulationsmechanismus mit dem die zelluläre Retinsäure‐Homöostase kontrolliert wird. (2) Ein frühzeitiger ROS‐Anstieg führt zu gezielten oxidativen Modifizierungen an Proteinen downstream des klassischen Retinsäure‐Signalweges. Zusammenfassung II Potenziell redox‐sensitive Proteine können dabei Teile von Signalkaskaden sein, die auf veränderte energetische Bedürfnisse der neuronal differenzierenden Zelle reagieren und zu der Ausbildung phänotypischer Merkmale beitragen können. Summary III Summary The human neuroblastoma cell line SH‐SY5Y is a common model system to study the initial phases of neuronal differentiation. Although the progression induced by retinoic acid is increasingly associated with redox‐sensitive processes, the molecular effects on proteins are not fully elucidated. Here, the influence of oxidative changes during the early stages of neuronal differentiation was investigated using proteome analysis. The applied differentiation protocol results in neurite outgrowth, a characteristic event of the neuronal differentiation process. Using quantitative mass spectrometry‐based proteomics changes in protein abundance were analyzed in a dynamic time course between 0 ‐ 48 h and after 120 h. In combination with quantitative mRNA data, changes in marker proteins involved in the retinoic acid signaling pathway (e.g. CRABP2) as well as neuronal differentiation (e.g. RET) were detected. Along with the largest morphological change within 48 h, proteome analyzes indicates a cellular change from proliferation to differentiation. Subsequently, experiments with redox‐sensitive sensors (RoGFP2) indicate a significant increase in cytosolic H2O2 after 2 h of retinoic acid treatment, probably involving the activity of the NADPH oxidase‐2 complex. A redox‐specific protein enrichment protocol was established to facilitate the global analysis of reversibly oxidized proteins by quantitative mass spectrometry. While only < 0.5% of the proteins showed a change in abundance between 0 ‐ 3 h, the analysis of the redox proteome revealed profound changes after 2 h of treatment. Out of 1 769 quantified cysteine containing proteins, 275 showed a significant difference between retinoic acid treated and control cells. Among these are proteins with known connections to redox‐sensitive processes and/or neurological development (e.g. RAC1). Furthermore, the results provided valuable hints about the reversibly oxidized cysteines for 57 of these 275 proteins. Finally, the addition of reducing agent N‐acetylcysteine or the free radical scavenger butylated hydroxyanisole let to a change in the cellular redox state, affected the abundance of marker proteins and reduced the neurite elongation. This experiment is another indication that the cellular redox state is important for the neuronal differentiation of the model. Based on the data obtained in this thesis, two models were proposed explaining the molecular effects of altered ROS levels. (1) ROS are part of the autoregulatory mechanism by which cellular retinoic acid homeostasis is controlled. (2) An early increase in ROS leads to targeted oxidative modifications of proteins downstream of the classical retinoic acid signaling pathway. Potentially redox‐sensitive proteins can be part of signaling cascades that react according to altered energetic needs of neuronal differentiating cells and contribute to phenotypic characteristics. Inhaltsverzeichnis IV Inhaltsverzeichnis Zusammenfassung I Summary III Abbildungsverzeichnis VII Tabellenverzeichnis VIII 1. Einleitung 1 1.1 Reaktive Spezies und oxidative Modifizierungen an Proteinen .................................................. 1 1.1.1 Cystein als Ziel oxidativer Modifizierungen .................................................................... 1 1.1.2 Reaktive Sauerstoffspezies .................................................................................................. 4 1.2 Quellen reaktiver Sauerstoffspezies ................................................................................................. 5 1.2.1 Mitochondriale Entstehung ................................................................................................. 6 1.2.2 Enzymatische Entstehung ................................................................................................... 7 1.2.3 Kontrolle des zellulären Redoxstatus ................................................................................ 9 1.3 Redox‐getriebene Prozesse während der neuronalen Differenzierung .................................... 11 1.3.1 Bi‐Funktionalität von reaktiven Sauerstoffspezies ......................................................... 13 1.4 Humane Neuroblastom‐Zelllinie SH‐SY5Y als Modellsystem ................................................... 14 1.4.1 Modellsystem zur Studie redox‐sensitiver Prozesse ...................................................... 14 1.4.2 Modellsystem zur Studie neuronaler Differenzierung .................................................. 16 1.4.3 Kultivierung und neuronale Differenzierung durch Retinsäure ................................. 16 1.5 Redox‐spezifische Analysen ............................................................................................................ 19 1.5.1 Kriterien für die redox‐spezifische Probenaufarbeitung ............................................... 20 1.5.2 Quantitative Thiolnachweise ............................................................................................ 22 1.5.3 Manipulation des zellulären Redoxzustandes ................................................................ 26 2. Zielsetzung der Arbeit 27 3. Material und Methoden 28 3.1 Chemikalien ....................................................................................................................................... 28 3.2 Molekularbiologische Methoden .................................................................................................... 28 3.2.1 Polymerase‐Kettenreaktion ..............................................................................................
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