Proteasome Inhibition Promotes Parkin-Ubc13 Interaction and Lysine 63-Linked Ubiquitination

Grace G Y Lim,Roy W X Sim,Xiao-Hui Ng,Katherine C M Chew,Kah-Leong Lim,Chou Chai,Jeanne M M Tan,Adeline Henry-Basil,Mark R Cookson

doi:10.1371/journal.pone.0073235

Abstract

Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination (which is usually uncoupled from the proteasome) may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired.

Highlights

The proteasome is a major intracellular proteolytic machinery that plays a vital role in maintaining cellular protein homeostasis through its ability to destroy unwanted proteins rapidly [1]
To test our hypothesis that parkin-mediated K63 ubiquitination may be enhanced in cells undergoing proteasomal stress, we examined the immunoreactivity of anti-UbK63 in Triton-X-100-soluble (S) and -insoluble (P) lysates sequentially prepared from parkin-expressing cells in the presence or absence of proteasome inhibition
Our study identifies a mechanism by which parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress, which appears to facilitate the subsequent clearance of selected parkin substrates via autophagy

Summary

Introduction

The proteasome is a major intracellular proteolytic machinery that plays a vital role in maintaining cellular protein homeostasis through its ability to destroy unwanted proteins rapidly [1]. It is noteworthy that the cell is capable of mediating alternative ubiquitin modifications such as monoubiquitination and K63-linked polyubiquitination whose roles are typically uncoupled from the proteasome [3]. We have previously hypothesized that non-proteolytic ubiquitination of proteins may help divert proteins destined for proteasomal degradation away from the system when it becomes overwhelmed under conditions of proteolytic stress [4]. The diverted proteins, which may aggregate into inclusion bodies, are acted upon by the complementary macroautophagy system (hereafter referred to as “autophagy”). In this way, the cell could preserve its proteasome function over prolonged periods of proteolytic stress and recover thereafter. Supporting our hypothesis, we have recently demonstrated that K63-linked polyubiquitination promotes the formation and autophagic clearance of protein inclusions [5,6]

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