title: The phosphorylation of Hsp20 enhances its association with amyloid-β to increase protection against neuronal cell death.
abstract: Up-regulation of Hsp20 protein levels in response to amyloid fibril formation is considered a key protective response against the onset of Alzheimer's disease (AD). Indeed, the physical interaction between Hsp20 and Aβ is known to prevent Aβ oligomerisation and protects neuronal cells from Aβ mediated toxicity, however, details of the molecular mechanism and regulatory cell signalling events behind this process have remained elusive. Using both conventional MTT end-point assays and novel real time measurement of cell impedance, we show that Hsp20 protects human neuroblastoma SH-SY5Y cells from the neurotoxic effects of Aβ. In an attempt to provide a mechanism for the neuroprotection afforded by Hsp20, we used peptide array, co-immunoprecipitation analysis and NMR techniques to map the interaction between Hsp20 and Aβ and report a binding mode where Hsp20 binds adjacent to the oligomerisation domain of Aβ, preventing aggregation. The Hsp20/Aβ interaction is enhanced by Hsp20 phosphorylation, which serves to increase association with low molecular weight Aβ species and decrease the effective concentration of Hsp20 required to disrupt the formation of amyloid oligomers. Finally, using a novel fluorescent assay for the real time evaluation of morphology-specific Aβ aggregation, we show that phospho-dependency of this effect is more pronounced for fibrils than for globular Aβ forms and that 25mers corresponding to the Hsp20 N-terminal can be used as Aβ aggregate inhibitors. Our report is the first to provide a molecular model for the Hsp20/Aβ complex and the first to suggest that modulation of the cAMP/cGMP pathways could be a novel route to enhance Hsp20-mediated attenuation of Aβ fibril neurotoxicity.
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Veterinary, Medical and life sciences, University of Glasgow, Glasgow G128QQ, UK.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Cameron
authors.affiliation: SUPA School of Physics and Astronomy, University of St Andrews, North Haugh, Fife KY169SS, UK.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Quinn
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Veterinary, Medical and life sciences, University of Glasgow, Glasgow G128QQ, UK.
authors.forename: Lynn S
authors.initials: LS
authors.lastname: Cairns
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Veterinary, Medical and life sciences, University of Glasgow, Glasgow G128QQ, UK.
authors.forename: Ruth
authors.initials: R
authors.lastname: MacLeod
authors.affiliation: SUPA School of Physics and Astronomy, University of St Andrews, North Haugh, Fife KY169SS, UK.
authors.forename: Ifor D W
authors.initials: ID
authors.lastname: Samuel
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Veterinary, Medical and life sciences, University of Glasgow, Glasgow G128QQ, UK.
authors.forename: Brian O
authors.initials: BO
authors.lastname: Smith
authors.affiliation: SUPA School of Physics and Astronomy, University of St Andrews, North Haugh, Fife KY169SS, UK.
authors.forename: J
authors.initials: J
authors.lastname: Carlos Penedo
authors.affiliation: Institute of Cardiovascular and Medical Science, College of Veterinary, Medical and life sciences, University of Glasgow, Glasgow G128QQ, UK. Electronic address: George.Baillie@glasgow.ac.uk.
authors.forename: George S
authors.initials: GS
authors.lastname: Baillie
mesh_terms: D016229:Amyloid beta-Peptides / Q000378:metabolism* / Q000494:pharmacology*; D001120:Arginine / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D050886:HSP20 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000473:pathology; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology*; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D013997:Time Factors
chemical_list: D016229:Amyloid beta-Peptides; D050886:HSP20 Heat-Shock Proteins; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D001120:Arginine
keywords: Aβ oligomerisation; Hsp20; Peptide array