Dokument: The Kinetics of α-Synuclein Aggregation: Single Fibril Growth, β1-β2 Contacts, Membrane Environments and Dityrosine Formation
| Titel: | The Kinetics of α-Synuclein Aggregation: Single Fibril Growth, β1-β2 Contacts, Membrane Environments and Dityrosine Formation | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=43418 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20170914-111133-1 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Wördehoff, Michael [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Willbold, Dieter [Gutachter] Dr. Hoyer, Wolfgang [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Parkinson’s disease (PD) is the second most common neurodegenerative disease. In PD, aggregation of the protein α-synuclein (α-syn) in the substantia nigra is the central hallmark of pathogenesis. Therefore, understanding the aggregation process itself as well as identifying factors that influence it is crucial. In this respect, this cumulative PhD thesis investigates the aggregation kinetics of α-syn in dependence of several factors in a comprehensive and detailed manner in four dedicated chapters.
In chapter one, the aggregation of α-syn is studied by high-resolution fluorescence microscopy. This in situ real-time analysis of aggregation lead to the detection of a stop-and-go mechanism of fibril growth, where growth phases were consistently interrupted by stop phases. Stop and growth phases were isoenergetic and interconverted with rate constants k+/− of 1.5 x 10−4 s−1. On average, α-syn fibrils grew with a growth rate of 8.5 nm min−1, which corresponded to a growth rate constant of 8.6 x 10−3 M−1 s−1. The development of this in situ assay enables the detailed study of the aggregation process and factors that influence it. Chapter two covers the influence of a covalent linkage of β1- and β2-fragments of α-syn on its aggregation and the aggregation of amyloid-β and IAPP. A covalent disulfide linkage of β1- and β2-fragments (called α-synCC) inhibited its aggregation, both de novo and seeded aggregation. α-SynCC also inhibited the aggregation of other amyloid proteins substoichio-metrically and greatly reduced cytotoxicity of α-syn, amyloid-β and IAPP in SH-S5Y5 cell culture. The identification of this inhibitory conformation that abolishes amyloid aggregate formation harbours great potential for the develepmont of therapeutic molecules. In chapter three, the effect of membrane-binding on α-syn aggregation is investigated, as α-syn is both a cytosolic and a membrane-associated protein. To mimick physiological lipid bilayers, we used nanodiscs (ND) composed of different lipids (different head group charge, fatty acid type, size) framed by a membrane scaffold protein. By using several biophysical methods we deciphered kinetic, thermodynamic and mechanistic properties of α-syn interaction with membranes, e.g. the higher the negative charge and available membrane surface area, the more α-syn residues were bound and aggregation was inhibited. In low membrane surface area conditions however, α-syn aggregation was stimulated. This study provided detailed, residue-resolved insights into membrane-binding of α-syn and concomitant aggregation behaviour. Chapter four deals with the influence of oxidative-stress-associated dityrosine (DiY)-crosslinking in α-syn, as increased oxidative stress is another hallmark of PD. We used UV-irradiation-assisted DiY formation as a model system to specifically form dityrosines. α-Syn as an intrinsically disordered protein showed a high tendency to form both intra- and intermolecular dityrosine crosslinks, independent from tyrosine position. Intermolecular DiY dimers strongly inhibited both de novo and seeded aggregation of wildtype α-syn substoichio-metrically. DiY-crosslinking of preformed α-syn aggregates stabilized them against dissolution, partly dissolved DiY-crosslinked aggregates acted as efficient on-pathway seeds for α-syn aggregation. This study showed that dityrosine formation can both inhibit aggregation but also stabilize toxic aggregates of α-syn. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie | |||||||
| Dokument erstellt am: | 14.09.2017 | |||||||
| Dateien geändert am: | 14.09.2017 | |||||||
| Promotionsantrag am: | 05.07.2017 | |||||||
| Datum der Promotion: | 08.08.2017 |
