Dokument: Selection and Characterization of Engineered Binding Proteins to a-Synuclein
| Titel: | Selection and Characterization of Engineered Binding Proteins to a-Synuclein | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=35974 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20151019-095444-2 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dr. Shaykhalishahi, Hamed [Autor] | |||||||
| Dateien: |
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| Beitragende: | Dr. Wolfgang Hoyer [Gutachter] Prof. Dr. Willbold, Dieter [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Amyloidogenesis or formation of amyloid fibrils is associated with more than 20 human diseases including Parkinson’s disease (PD), Alzheimer’s disease (AD), and type 2 diabetes (T2D). PD is characterized by brain deposition of Lewy bodies, mainly composed of a-synuclein fibrils. Increasing evidence establishes the involvement of a-synuclein aggregation in the pathogenesis of PD. Despite extensive research on protein aggregation, the exact mechanisms controlling or triggering this process remain unknown. Identification of specific binding proteins may provide a precious tool for the study of protein-protein interactions in the aggregation process. Previous studies using a selected binding protein against amyloid-b (Ab) peptide, ZAb3, resulted in the identification of a b-hairpin-forming region critical for Ab aggregation. The overall aim of the present thesis is focused on the selection of engineered binding proteins for a-synuclein and characterization of their interactions with a-synuclein and other amyloidogenic proteins. Present thesis describes the selection of AS69, a specific b-wrapin protein with nanomolar affinity to -synuclein. Similar to ZAb3, dimeric AS69 forms a hydrophobic tunnel-like cavity in which a-synuclein adopts a b-hairpin conformation. The b-hairpin structure is composed of two b-strands which are similar to those identified in the fibril core. Location of these b-strands in the core of a-synuclein oligomers and the presence of 5 familial mutations linked to early-onset PD highlight the importance of the identified b-hairpin forming region. Here, it is shown that sequestration of the b-hairpin structure inside of AS69 impedes a-synuclein aggregation and toxicity. Remarkably, sub-stoichiometric amounts of AS69 prolong lag time of aggregation via interference with primary and/or secondary nucleation events. Out of set of selected b-wrapin proteins, AS10 interacts not only with a-synuclein but also with Ab and Islet amyloid polypeptide (IAPP) with sub-micromolar affinity. Our NMR data demonstrate that AS10 binding is accompanied by the formation of b-hairpin structure in all three target proteins. These results emphasize the similarity of these amyloidogenic proteins with respect to conformational properties and molecular recognition motifs. AS69 is a homodimer protein linked via Cys-28 residues of the subunits. Employing AS69-GS3, the head-to-tail construct of dimeric AS69, construct reveals the importance of Cys-28 for the binding affinity to a-synuclein. Our biophysical data demonstrate an increase in structural compaction and stability induced by formation of interamolecular Cys-28 linkage. In next study, b-hairpin region identified by AS69 is further elucidated using a-synCC, a double-cysteine mutant of a-synuclein. Here, stabilization of contact between the b1 and b2 segments of a-synuclein via introduction of an intramolecular disulfide bond leads to the loss of aggregation propensity of a-synuclein. Our results also demonstrate the inhibitory effect of a-synCC on the fibrillation process of a-synuclein, Ab and IAPP. Taken together, we propose that the b1-b2 contact in a-synuclein entails an inhibitory effect against amyloid formation and that disulfide bond-assisted stabilization of this contact in a-synCC strengthens this effect. This thesis demonstrates how engineered binding proteins can be applied to (i) identify sequence regions critical for protein aggregation, (ii) elucidate common interaction motifs of intrinsically disordered amyloidogenic proteins, (iii) devise novel therapeutic strategies for protein misfolding disorders. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 19.10.2015 | |||||||
| Dateien geändert am: | 19.10.2015 | |||||||
| Promotionsantrag am: | 03.11.2014 | |||||||
| Datum der Promotion: | 25.11.2014 |
