title: Ubiquitinylation of α-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is regulated by Bcl-2-associated athanogene 5 (BAG5).
abstract: Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization.
authors.affiliation: Department of Neurology, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States of America. lorraine.kalia@utoronto.ca
authors.forename: Lorraine V
authors.initials: LV
authors.lastname: Kalia
authors.forename: Suneil K
authors.initials: SK
authors.lastname: Kalia
authors.forename: Hien
authors.initials: H
authors.lastname: Chau
authors.forename: Andres M
authors.initials: AM
authors.lastname: Lozano
authors.forename: Bradley T
authors.initials: BT
authors.lastname: Hyman
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: McLean
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000368:Aged; D000369:Aged, 80 and over; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D005260:Female; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D054875:Ubiquitination; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C496515:BAG5 protein, human; D002352:Carrier Proteins; D051844:alpha-Synuclein; D008156:Luciferases; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases