Ubiquitinylation of α-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein alpha-synuclein (alpha-syn). Mutations within or multiplications of the gene encoding alpha-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. alpha-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 alpha-syn can self associate into soluble oligomeric species and implicates these alpha-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 alpha-syn oligomers. Here we demonstrate that alpha-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 alpha-syn. We also demonstrate that CHIP and alpha-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 alpha-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of alpha-syn oligomerization to investigate regulation of alpha-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce alpha-syn oligomerization and that non-ubiquitinylated alpha-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of alpha-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of alpha-syn oligomerization.