N-homocysteinylation of ovine prion protein induces amyloid-like transformation.
Modification of protein lysyl residues by homocysteine (Hcy)-thiolactone generates proteins with altered structures and functions. It has been supposed to be one of the factors inducing protein condensation pathologies. To test a hypothesis that N-homocysteinylation may induce structural changes and in particular amyloidogenic conversion, ovine prion protein (PrP) was modified with Hcy-thiolactone and its physico-chemical properties were studied. N-Hcy-PrP formed insoluble multimers. Mass spectrometry analyses showed that at least K197 and K207 residues of PrP were the sites of N-homocysteinylation. Dynamic light scattering measurements revealed large aggregated N-Hcy-PrP particles of 1μm diameter. They were resistant to proteinase K digestion, and enhanced thioflavin T (ThT)-binding fluorescence, what is characteristic of amyloid structures. Infrared spectroscopy measurements showed increased content of beta-sheet in N-Hcy-PrP compared to unmodified PrP. Epifluorescence microscopy in the presence of ThT revealed cluster-like aggregates of N-Hcy-PrP. The collected data indicate that the N-homocysteinylation causes amyloidogenic transformation of PrP in vitro.
UR 1268 Biopolymères Interactions Assemblages, INRA, équipe Fonctions et Interactions des Protéines, Nantes Cedex, France.
Yulia Y
YY
Stroylova
Jean-Marc
JM
Chobert
Vladimir I
VI
Muronetz
Hieronim
H
Jakubowski
Thomas
T
Haertlé
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D019286:Endopeptidase K / Q000378:metabolism; D006710:Homocysteine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006868:Hydrolysis / Q000187:drug effects; D007783:Lactones / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects*; D017433:Protein Structure, Secondary / Q000187:drug effects; D012756:Sheep
D000682:Amyloid; D007783:Lactones; D011328:Prions; D006710:Homocysteine; D019286:Endopeptidase K