Dokument: Characterization of the human prion protein in its native-like state
| Titel: | Characterization of the human prion protein in its native-like state | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=62023 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20240321-101705-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Bolakhrif, Najoua [Autor] | |||||||
| Dateien: |
| |||||||
| Beitragende: | Prof.Dr. Willbold Dieter [Gutachter] Dr. Hoyer, Wolfgang [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The human prion protein (huPrP) is a membrane-bound glycoprotein found mainly in the nervous system. HuPrP in its native conformation (huPrPC) contains a globular C-terminal domain rich in α-helical structure and an unstructured N-terminal domain. Its physiological function has not been fully elucidated. HuPrPC can undergo structural conversion to an infectious form (huPrPSc) rich in β-sheets typical for amyloid structure. This misfolded PrPSc is associated with prion diseases known as transmissible spongiform encephalopathies (TSEs). Susceptibility to TSE in humans is strongly influenced by a polymorphism of huPrP at position 129, which involves either valine (V) or methionine (M). In this work, different pathways of amyloid formation are presented for the 129V and 129M full-length variants of huPrP. Amyloid formation of the 129V variant starts directly from a destabilized monomeric state. The 129M variant undergoes a more complex aggregation process with oligomeric intermediates. The higher propensity of the 129M variant to form oligomers is already present prior to amyloid formation. A shorter construct of the 129M variant containing only the structured C-terminal domain lacks most oligomers and consequently exhibits similar aggregation behavior to the full-length 129V variant. This highlights the importance of the flexible N-terminus, which tends to go unnoticed in prion research, and emphasizes a possible interaction with the globular domain depending on the 129M/V polymorphism. These results contribute to the understanding of the differences between the two variants of the polymorphism at position 129 and a possible interaction between C- and N-terminus that might be related to the differences observed in pathology. To approach huPrP studies on a close to physiological level, an eukaryotic expression system was used to provide native-like full-length huPrP with all major post-translational modifications, including glycosylphosphatidylinositol-anchoring and mammalian-like glycosylation. Leishmania tarentolae (L. tarentolae) was used for stable and high-level expression of native-like huPrP. A purification protocol was established, involving the incorporation of native-like huPrP into micelles. The purified native-like huPrP possessed a conformation dominated by α-helical and random coil structure and was predominantly monomeric (∼90 %). Comparison of native-like huPrP (expressed in L. tarentolae) and huPrP (expressed in Escherichia coli (E. coli)) revealed differences in secondary structure and solubility at physiological conditions. HuPrP expressed in E. coli is less soluble at pH 7.4, containing a majority of aggregates (∼71 %), while the remaining protein was mainly monomeric (∼22 %). The soluble fraction of huPrP expressed in E. coli indicates an increased amount of β-sheet structure. The purified native-like huPrP expressed in L. tarentolae is superior to huPrP expressed in E. coli for studies under physiological conditions and holds great potential for studies focusing on high-resolution structures or kinetics of misfolding. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie | |||||||
| Dokument erstellt am: | 21.03.2024 | |||||||
| Dateien geändert am: | 21.03.2024 | |||||||
| Promotionsantrag am: | 03.01.2023 | |||||||
| Datum der Promotion: | 27.01.2023 |
