title: Methionine oxidation accelerates the aggregation and enhances the neurotoxicity of the D178N variant of the human prion protein.
abstract: The D178N mutation of the prion protein (PrP) results in the hereditary prion disease fatal familial insomnia (FFI). Little is known regarding the effects of methionine oxidation on the pathogenesis of D178N-associated FFI. In the present study, we found that the D178N variant was more susceptible to oxidation than wild-type PrP, as indicated by reverse-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) analysis. Circular dichroism (CD), differential scanning calorimetry (DSC), thioflavin T (ThT) binding assay studies demonstrated that methionine oxidation decreased the structural stability of the D178N variant, and the oxidized D178N variant exhibited a greater propensity to form β-sheet-rich oligomers and aggregates. Moreover, these aggregates of oxidized D178N PrP were more resistant to proteinase K (PK) digestion. Additionally, using fluorescence confocal microscopy, we detected a high degree of aggregation in D178N-transfected Neuro-2a (N2a) cells after treatment with hydrogen peroxide (H2O2). Furthermore, the oxidation and consequent aggregation of the D178N variant induced greater apoptosis of N2a cells, as monitored using flow cytometry. Collectively, these observations suggest that methionine oxidation accelerates the aggregation and enhances the neurotoxicity of the D178N variant, possibly providing direct evidence to link the pathogenesis of D178N-associated FFI with methionine oxidation.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Boya
authors.initials: B
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Zonglin
authors.initials: Z
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Jin
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Shaobo
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China. Electronic address: xiaogf@wh.iov.cn.
authors.forename: Gengfu
authors.initials: G
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; University of the Chinese Academy of Sciences, Beijing 100039, China. Electronic address: Zhouzheng@wh.iov.cn.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019286:Endopeptidase K / Q000378:metabolism; D020022:Genetic Predisposition to Disease / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D034062:Insomnia, Fatal Familial / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008715:Methionine / Q000378:metabolism*; D018613:Microscopy, Confocal; D020125:Mutation, Missense; D020258:Neurotoxicity Syndromes / Q000235:genetics* / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D011328:Prions / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects
chemical_list: D016877:Oxidants; D011328:Prions; D008715:Methionine; D006861:Hydrogen Peroxide; D019286:Endopeptidase K
keywords: Aggregates; Apoptosis; D178N mutation; Fatal familial insomnia; Oxidative stress