title: Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.
abstract: Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27.
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Blagojce
authors.initials: B
authors.lastname: Jovcevski
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Kelly
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Anthea P
authors.initials: AP
authors.lastname: Rote
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Tracey
authors.initials: T
authors.lastname: Berg
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Heidi Y
authors.initials: HY
authors.lastname: Gastall
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Justin L P
authors.initials: JL
authors.lastname: Benesch
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. Electronic address: aquilina@uow.edu.au.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Aquilina
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. Electronic address: heathe@uow.edu.au.
authors.forename: Heath
authors.initials: H
authors.lastname: Ecroyd
mesh_terms: D002460:Cell Line; D002942:Circular Dichroism; D019281:Dimerization; D057809:HEK293 Cells; D055551:HSP27 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D055551:HSP27 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins