title: Cyclin-dependent kinase 5 phosphorylation of familial prion protein mutants exacerbates conversion into amyloid structure.
abstract: Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion.
authors.affiliation: From the Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Department of Neurology and Neurosurgery, McGill University, Montréal, Québec H3T 1E2, Canada and.
authors.forename: Raphaël
authors.initials: R
authors.lastname: Rouget
authors.affiliation: From the Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Department of Neurology and Neurosurgery, McGill University, Montréal, Québec H3T 1E2, Canada and Department of Neurology and Neurosurgery, McGill University, 3775 University Street, Montréal, Québec H3A 2B4, Canada.
authors.forename: Gyanesh
authors.initials: G
authors.lastname: Sharma
authors.affiliation: From the Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Department of Neurology and Neurosurgery, McGill University, Montréal, Québec H3T 1E2, Canada and Department of Neurology and Neurosurgery, McGill University, 3775 University Street, Montréal, Québec H3A 2B4, Canada andrea.leblanc@mcgill.ca.
authors.forename: Andréa C
authors.initials: AC
authors.lastname: LeBlanc
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D052160:Benzothiazoles; D015153:Blotting, Western; D002942:Circular Dichroism; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D010766:Phosphorylation; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D011328:Prions; D013844:Thiazoles; C009462:thioflavin T; D012694:Serine; D051360:Cyclin-Dependent Kinase 5
keywords: Amyloid; Circular Dichroism (CD); Phosphorylation; Prion; Prion Disease