CHIP Targets Toxic α-Synuclein Oligomers for Degradation

Julie E Tetzlaff,Preeti Putcha,Tiago F Outeiro,Alexander Ivanov,Oksana Berezovska,Bradley T Hyman,Pamela J Mclean

doi:10.1074/jbc.m802283200

Abstract

alpha-Synuclein (alphaSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric alphaSyn intermediates may represent the most toxic alphaSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on alphaSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced alphaSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic alphaSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of alphaSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric alphaSyn may contribute to the successful development of drug therapies that target oligomeric alphaSyn by mimicking or enhancing the powerful effects of CHIP.

Highlights

Molecular chaperones such as heat shock proteins (Hsp) 70 and 90 and their associated co-chaperone CHIP have a well documented ability to reduce toxicity mediated by neurodegenerative disease states, stress, or injury (8 –17)
To further investigate the specific ␣Syn species that is targeted by CHIP, we employed a novel bimolecular fluorescence complementation (BiFC) assay that we have previously demonstrated to be a specific and effective method by which to visualize and monitor ␣Syn oligomerization in vitro [19]
We investigated the ability of CHIP to reduce ␣Syn oligomerization by transfecting H4 human neuroglioma cells with the ␣Syn BiFC constructs ␣SynGC and GN-␣Syn in the presence or absence of CHIP (Fig. 1) and found that CHIP dramatically reduced ␣Syn oligomerization as measured by intensity of GFP fluorescence (Fig. 2, A and B)

Summary

Introduction

Molecular chaperones such as heat shock proteins (Hsp) 70 and 90 and their associated co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein) have a well documented ability to reduce toxicity mediated by neurodegenerative disease states, stress, or injury (8 –17). To confirm that the effect of CHIP on ␣Syn oligomerization is specific, we overexpressed domain mutant constructs of CHIP (CHIP⌬U and CHIP⌬TPR) with ␣SynGC and GN-␣Syn (Fig. 2C) and determined that CHIP⌬U, but not CHIP⌬TPR, was able to significantly reduce the fluorescence produced by ␣Syn oligomer formation (Fig. 2D). Because the TPR domain of CHIP is essential for Hsp70 interactions, these data suggest that the CHIP-mediated reduction in ␣Syn oligomer formation occurs in an Hsp70-associated manner.

Results

Conclusion