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Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-5582-140X
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
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
Abstract [en]

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.
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-53174DOI: 10.1074/jbc.M112.372136ISI: 000306916300025OAI: 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||

Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2014-04-08
In thesis
1. Early events in disease associated protein misfolding
Open this publication in new window or tab >>Early events in disease associated protein misfolding
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The scope of this thesis is to unravel some of the mysteries concerning events takingplace early in the amyloid cascade. In vitro studies of early misfolded states ofamyloidogenic proteins are important since the use of recombinant proteins allow us to monitor slight changes in environmental conditions as well as in amino acid composition and thereby illuminate the problem at near atomic resolution.

Human prion protein (HuPrP) (associated with e.g. Creutzfeldt-Jakob disease) andthe Aβ1-42 peptide (associated with Alzheimer’s disease) recombinantly expressed in Escherichia coli have been used as model systems for these studies.

A new protocol for amyloid fibril formation of human prion protein under native conditions was developed. This revealed an unusual pathway of conformational conversion from early formed disordered aggregates that later matured into amyloidfibrils.

The polymorphism 129M/V in HuPrP has a large impact on susceptibility both to sporadic and infectious prion diseases. Some features of this polymorphism havebeen elucidated, employing a mutational study in position 129 (M, A, L, V, P, M, W,E, and K). These investigations have rendered new knowledge about the impact ofsize, charge and β-carbon branching in position 129 upon early intermolecular interactions and the effects of fibril seeding.

Investigations of the interactions between different assembly forms of HuPrP and components of the innate immune system revealed that both native, oligomeric and fibrillar forms of HuPrP activate both the classical and alternative pathways of the Complement System. Most efficient activation is achieved upon binding of oligomeric HuPrP to the complement component C1q.

We have developed a system for recombinant expression of human A,1-42. The monomeric peptides are assembled into various sized soluble oligomers (trimer, hexamer, nonamer, dodecamer). The oligomeric forms were stable in 8 M urea, 6 MGuHCl and SDS suggesting that these were covalently cross-linked. Some mechanistic features in the assembly process have been investigated and we have shown that cupric ions facilitates formation of stable oligomers in our system.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 116 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1270
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:liu:diva-52743 (URN)978-91-7393-544-9 (ISBN)
Public defence
2009-11-20, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Available from: 2010-01-18 Created: 2010-01-12 Last updated: 2012-11-15Bibliographically approved

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