Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 31, 25975-25984 p.Article in journal (Refereed) Published
The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met129 (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90–231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, Tm = 65–66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3–4 °C. The most destabilizing substitution was M129P, which lowered the Tm by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.
Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology , 2012. Vol. 287, no 31, 25975-25984 p.
IdentifiersURN: urn:nbn:se:liu:diva-53174DOI: 10.1074/jbc.M112.372136ISI: 000306916300025OAI: oai:DiVA.org:liu-53174DiVA: diva2:287403
funding agencies|EU-FP7 Health Programme Project LUPAS||Swedish Research Council||Knut and Alice Wallenberg Foundation||Swedish Foundation for Strategic Research||Linkoping University Center for Neuroscience||2010-01-182010-01-182014-04-08