Dokument: Hell is other polypeptides - Studying the role of β-hairpins in amyloid formation

Titel:	Hell is other polypeptides - Studying the role of β-hairpins in amyloid formation
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=55067
URN (NBN):	urn:nbn:de:hbz:061-20211229-084653-6
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Agerschou, Emil Dandanell [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 46,16 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 16.12.2020 / geändert 16.12.2020
Beitragende:	Prof. Dr. Hoyer, Wolfgang [Gutachter] Prof. Dr. Büll, Alexander Kai [Gutachter]
Stichwörter:	biophysics, kinetics, amyloid, α-synuclein, β-hairpin
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	Amyloid fibrils are highly ordered insoluble protein aggregates wherein the protein adopts the cross-β conformation. Very dissimilar sequences can adopt this conformation and aggregate, which is often seen in neurodegenerative diseases. In Parkinson’s disease, amyloid fibrils of α-synuclein (αS) accumulate. In this thesis, the mechanisms of amyloid formation and inhibition of αS are studied. This is done mainly by elucidating the inhibition mechanisms of previously identified inhibitors. The region of αS composed of residues 37-54 has previously been shown to adopt a β-hairpin when bound to an engineered binding protein known as AS69. Many of the known disease asso- ciated mutations are found within this region. Furthermore, binding of AS69 inhibits conversion of soluble αS into amyloid fibril in a highly substoichiometric manner, however, the mechanism was not understood. Here, the mechanism of inhibition was investigated by selectively favouring specific amyloid-forming pathways by tuning the solution conditions. It was found that AS69 only had a stoichiometric effect on elongation, compatible with sequestration of textalpha S monomers by AS69. Secondary nucleation was found to be highly substoichiometrically inhib- ited. By linking AS69 to αS, it was shown that inhibition of secondary nucleation likely was caused by the complex of AS69 and αS rather than by AS69 alone. Earlier, favouring the β-hairpin conformation of αS was done by introducing a disulphide bond between residue 41 and 48 using cysteine mutations (CC48). CC48 did not form amyloids unless the disulphide bond was reduced. Furthermore, CC48 inhibited elongation of wild-type (WT) fibrils. Here, the position dependency of the disulphide bond was studied by mutations. A large variation in inhibition strength was seen among these mutants, but, CC48 was the strongest inhibitor. Applying the theoretical framework from reversible enzyme inhibition, it was established that CC48 caused inhibition by competing with WT for binding to the fibril- end. However, an increase in efficiency of inhibition at high WT concentrations was observed. Modelling of this unusual behaviour where the substrate, WT, cooperated with the inhibitor, CC48, to accomplish inhibition revealed that two additional WT monomers could bind to fibril-ends where CC48 was already bound. The proposed mechanism was corroborated by constructing linked dimers of WT and CC48 which exhibited substantially higher inhibition efficiency than monomeric CC48. An alternative to stabilisation of the β-hairpin conformation by disulphide bonds, was stabilising it by increased turn-formation propensity. αS mutants with increased β-hairpin propensity, through stabilised β-turn propensity, were prepared. Here, the ability of these mutants to form amyloid was evaluated and, surprisingly, the mutants tended to have increased aggregation kinetics compared to the WT. Lastly, cross-elongation experiments revealed a large asymmetry between ability to elongate non-self fibrils, and ability to be elongated by non-self monomers. β-Hairpin formation upon binding to inhibitors has been observed for several amyloidogenic proteins in addition to αS. A single wrapin, AS10, binds, induces a β-hairpin, and inhibits three different amyloidogenic proteins. Here, the human proteome was analysed to locate potentially amyloidogenic sequences that would bind AS10. The TANGO algorithm was used as the firstfilter, and to increase specificity, biophysical characteristics of known AS10 binders were used as the second filter. A surprisingly large number of sequences were located. Interactions of proteins, either among identical species to form amyloid or between different species causing inhibition, was the core subject of this thesis. The behaviour of individual polypeptides, like people, was dominated by the presences of others. It thus seems that Sarte’s concept of "Hell is other people" might apply to polypeptides as well.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie
Dokument erstellt am:	29.12.2021
Dateien geändert am:	29.12.2021
Promotionsantrag am:	28.07.2020
Datum der Promotion:	07.12.2020