title: Cell-free analysis of polyQ-dependent protein aggregation and its inhibition by chaperone proteins.
abstract: Protein misfolding and aggregation is one of the major causes of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. So far protein aggregation related to these diseases has been studied using animals, cultured cells or purified proteins. In this study, we show that a newly synthesized polyglutamine protein implicated in Huntington's disease forms large aggregates in HeLa cells, and successfully recapitulate the process of this aggregation using a translation-based system derived from HeLa cell extracts. When the cell-free translation system was pre-incubated with recombinant human cytosolic chaperonin CCT, or the Hsc70 chaperone system (Hsc70s: Hsc70, Hsp40, and Hsp110), aggregate formation was inhibited in a dose-dependent manner. In contrast, when these chaperone proteins were added in a post-translational manner, aggregation was not prevented. These data led us to suggest that chaperonin CCT and Hsc70s interact with nascent polyglutamine proteins co-translationally or immediately after their synthesis in a fashion that prevents intra- and intermolecular interactions of aggregation-prone polyglutamine proteins. We conclude that the in vitro approach described here can be usefully employed to analyze the mechanisms that provoke polyglutamine-driven protein aggregation and to screen for molecules to prevent it.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan; RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan. Electronic address: machida@eng.u-hyogo.ac.jp.
authors.forename: Kodai
authors.initials: K
authors.lastname: Machida
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Shigeta
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Kobayashi
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Masumoto
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Yuna
authors.initials: Y
authors.lastname: Hidaka
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan; RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan. Electronic address: imataka@eng.u-hyogo.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Imataka
mesh_terms: D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D066329:Protein Aggregates / Q000187:drug effects*
chemical_list: D018832:Molecular Chaperones; D010455:Peptides; D066329:Protein Aggregates; C097188:polyglutamine
keywords: Aggregation; CCT; Cell-free; Chaperone; In vitro translation; Polyglutamine