β-Synuclein Reduces Proteasomal Inhibition by α-Synuclein but Not γ-Synuclein

Andrei Surguchov,Barry Festoff,Andreas Matouschek,Benjamin Wolozin,Kwame Mensah,Makoto Hashimoto,Eliazer Masliah,Cindy Hsu,Heather Snyder,Irina G. Surgucheva

doi:10.1074/jbc.m412887200

Abstract

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.

Highlights

The accumulation of aggregated ␣-synuclein is thought to contribute to the pathogenesis of Parkinson’s disease
␤-Synuclein Reduces Proteasomal Inhibition by ␣-Synuclein substantia nigra from subjects who died with Parkinson’s disease (PD), one study failed to detect changes in proteasomal activity in brain tissue from subjects who suffered from PD [13,14,15]
We examined the activity of 20 S proteasome in the presence of increasing concentrations of monomeric ␣-synuclein using synthetic fluorogenic peptides to monitor proteasomal activity

Summary

Introduction

The accumulation of aggregated ␣-synuclein is thought to contribute to the pathogenesis of Parkinson’s disease. One of the functions of the 19 S cap is to recognize ubiquitinated proteins, which is mediated by a protein in the 19 S cap, termed S6Ј [6] Aggregated proteins, such as those that accumulate as inclusion bodies in the brains of individuals with neurodegenerative disease, appear to potently inhibit proteasomal activity [7]. Polyglutamine repeat proteins, such as those that occur with Huntington’s disease, appear to be potent inhibitors of ubiquitin-dependent proteasomal function in cell culture [7]. These studies highlight the potential importance of the proteasome in the pathophysiology of PD

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Conclusion