The C289G and C418R missense mutations cause rapid sequestration of human Parkin into insoluble aggregates

Abstract

Mutations in the parkin gene are responsible for autosomal recessive parkinsonism. The disease-linked missense mutations are highly concentrated in the RING–IBR–RING domains of Parkin. In this study, we investigated the consequences of several missense parkin gene mutations in cell culture. We have demonstrated that two of these mutations (C289G and C418R), which replace consensus cysteine residues in the RING domains, significantly decrease the solubility of Parkin in cells. Upon overexpression, the presumably misfolded proteins formed cytoplasmic aggregates that concentrated into large perinuclear inclusion bodies when proteasome activity was inhibited. This process required active microtubule-dependent retrograde transport, as previously reported for aggresome formation. These results provide information on the molecular basis of the loss of function caused by mutations of critical residues in Parkin. They also contribute to our understanding of the cellular mechanism underlying the aggregation of mutant Parkin.

Introduction

Parkinson's disease is a common neurodegenerative disorder characterized by the massive loss of dopaminergic neurons and the presence of Lewy bodies in specific regions of the pars compacta of the substantia nigra (Fearnley and Lees, 1991). Parkin, one of several genes recently identified as responsible for monogenic forms of parkinsonism, causes an autosomal recessive form of the disease with early onset in most cases (Kitada et al., 1998). A variety of mutations and deletions in the parkin gene have been found in a large proportion of families with autosomal recessive parkinsonism Hattori et al., 1998, Abbas et al., 1999, Lücking et al., 2000. Missense mutations are largely found in the carboxyl terminal portion of the Parkin protein which consists of two RING (really interesting new gene) domains, an IBR (in between RING) domain, and a PDZ (postsynaptic density-95, disc large, zona occludens) domain Morett and Bork, 1999, Fallon et al., 2002. The consensus residues of the RING, IBR, and PDZ domains are identical in humans, rat, and mouse Gu et al., 2000, Fallon et al., 2002. The RING and IBR domains are essential for Parkin's E3 ubiquitin–protein ligase activity. This activity is responsible for the ubiquitylation of specific substrates in association with the E2 ubiquitin-conjugating enzymes UbcH7 or UbcH8 Shimura et al., 2000, Shimura et al., 2001, Imai et al., 2000, Imai et al., 2001, Zhang et al., 2000, Chung et al., 2001. Loss of this function is thought to cause the specific degeneration of dopaminergic neurons in parkinsonism due to parkin gene mutations.

Parkin is widely distributed throughout the human brain Shimura et al., 1999, Zarate-Lagunes et al., 2001, but was undetectable in the brain of a patient with a deletion in exon 4 of the parkin gene (Shimura et al., 1999), suggesting that the mutant protein is rapidly degraded. Deletions and missense mutations cause modifications of the primary structure of proteins and the formation of nonnative, misfolded intermediate products (Anfinsen, 1973) that are detected by endoplasmic reticulum quality control mechanisms (Ellgaard and Helenius, 2001). Nonnative proteins that cannot readily fold into the native form are removed by proteolysis or aggregation into aberrant subcellular compartments (Kopito, 2000). The ubiquitin–proteasome pathway is the major proteolytic system for degrading cytosolic, secretory, and membrane proteins (Kopito, 2000). Ubiquitylation of the substrate involves the sequential activity of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin–protein ligase (Hershko and Ciechanover, 1998). The ubiquitylated substrate is degraded into short peptides by the 26S proteasome which consists of the 20S proteasome, a cylindrical catalytic core protein, and two 19S regulatory caps that recognize the polyubiquitin chains on the substrates (Voges et al., 1999). Aggregation results from specific interactions between nonnative intermediate products (Speed et al., 1996). Cytoplasmic aggregates are formed by ordered polymerization around a seed (Lansbury, 1997). More ordered aggregation requires the microtubules and intermediate filaments of the intact cytoskeleton Johnston et al., 1998, Garcia-Mata et al., 1999.

Here, we have investigated the consequences of missense mutations affecting critical domains of the Parkin protein in families with autosomal recessive parkinsonism. Overexpression of the C289G and C418R Parkin mutants induced accumulation of presumably misfolded, aggregated proteins. These proteins tended to accumulate spontaneously in the perinuclear region, leading to formation of large aggresome-like inclusion bodies upon inhibition of proteasome activity.