Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein aggregation

Fanjun Meng,Dongdong Yao,Stuart A Lipton,Zezong Gu,Bernd Moosmann,Yuliang Ma,Eliezer Masliah,Joshua Reicher,Wei Wu,Yang Shi,Jonathan Kabakoff

doi:10.1186/1750-1326-6-34

Abstract

BackgroundAccumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson's disease (PD). Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death.ResultsTo gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation) and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP+), representing an in vitro cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H2O2 induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H2O2 accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS) analysis revealed that H2O2 reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs.ConclusionThese findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation.

Highlights

Accumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson’s disease (PD)
Using the antibody generated, we initially examined putative sulfonation of a number of cysteine-containing proteins after oxidative stress engendered by H2O2 in vitro
Our direct detection of sulfonated derivatives and subsequent aggregation of parkin in cell-based PD model, an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors and in PD brains, in conjunction with finding that parkin function is regulated by ROS, yields mechanistic insight into the chemical reactions of parkin under oxidative stress and their effect on ubiquitinproteasome system (UPS) impairment

Summary

Introduction

Accumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson’s disease (PD). Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death. Increasing evidence indicates that there may be a link between oxidative/nitrosative stress induced by reactive oxygen/nitrogen species (ROS/RNS) and accumulation of aberrant or misfolded proteins associated with ubiquitinproteasome system (UPS) dysfunction [11,12,13,14,15]. This cellular process involves tagging molecules targeted for degradation with polyubiquitin chains through a series of reactions carried out by ubiquitin enzymes. Dysfunction of parkin results in abnormal ubiquitination and accumulation of these substrates, which may contribute to cytoplasmic inclusion formation, impairment in UPS activity, and the demise of DA neurons [17,18]

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