Ubiquitinylation of α-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is regulated by Bcl-2-associated athanogene 5 (BAG5).

Lorraine V Kalia,Andres M Lozano,Suneil K Kalia,Hien Chau,Pamela J Mclean,Bradley T Hyman,Charleen T Chu

doi:10.1371/journal.pone.0014695

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.

Highlights

Parkinson’s disease (PD) is a movement disorder affecting approximately three percent of the population over the age of sixty-five and is second only to Alzheimer’s disease as the most common neurodegenerative disease [1]
bcl-2-associated athanogene 5 (BAG5) Inhibits the Ubiquitinylation of a-Synuclein by carboxyl terminus of Hsp70-interacting protein (CHIP) To begin to investigate the consequences of the CHIP-BAG5 association on CHIP function, we examined whether BAG5 affects the E3 ubiquitin ligase activity of CHIP
There is growing evidence that a subpopulation of a-syn may be covalently modified by ubiquitinylation and that ubiquitinylated asyn is present in Lewy bodies [44,45,46]

Summary

Introduction

Parkinson’s disease (PD) is a movement disorder affecting approximately three percent of the population over the age of sixty-five and is second only to Alzheimer’s disease as the most common neurodegenerative disease [1]. Idiopathic PD and most familial forms of PD are characterized by the presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons. A-syn as well as other proteins within Lewy bodies are frequently ubiquitinylated [2,3]. These inclusions contain members of the heat shock protein (Hsp) family such as Hsp70 [7,8,9], and co-chaperone molecules including carboxyl terminus of Hsp70-interacting protein (CHIP) [10] and bcl-2-associated athanogene 5 (BAG5) [11]

Methods

Results

Conclusion