Parkin mediates the degradation-independent ubiquitination of Hsp70

Mutations in the parkin gene cause autosomal recessive, juvenile-onset parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. Disease-associated mutations cause a loss-of-function of parkin which may compromise the poly-ubiquitination and proteasomal degradation of specific protein substrates, potentially leading to their deleterious accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro and in cultured cells. Parkin interacts with Hsp70 via its second RING finger domain and mutations in/near this domain compromise Hsp70 ubiquitination. Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor does it promote its proteasomal degradation. Consistent with this observation, Hsp70 levels remain unaltered in brains from parkin-deficient autosomal recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70 levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's disease/dementia with Lewy bodies brains. Parkin mediates the multiple mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent role for this ubiquitin modification. Our observations support a novel functional relationship between parkin and Hsc/Hsp70 and support the notion that parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins either via attachment of alternatively linked poly-ubiquitin chains or through multiple mono-ubiquitination to achieve alternate biological outcomes.     

BAG5 inhibits parkin and enhances dopaminergic neuron degeneration

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). Decreases in parkin activity may also contribute to neurodegeneration in sporadic forms of PD. Here, we show that bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacts with parkin and the chaperone Hsp70. Within this complex, BAG5 inhibits both parkin E3 ubiquitin ligase activity and Hsp70-mediated refolding of misfolded proteins. BAG5 enhances parkin sequestration within protein aggregates and mitigates parkin-dependent preservation of proteasome function. Finally, BAG5 enhances dopamine neuron death in an in vivo model of PD, whereas a mutant that inhibits BAG5 activity attenuates dopaminergic neurodegeneration. This contrasts with the antideath functions ascribed to BAG family members and suggests a potential role for BAG5 in promoting neurodegeneration in sporadic PD through its functional interactions with parkin and Hsp70.     

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.     

Chaperone functions of the E3 ubiquitin ligase CHIP

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone as well as an E3 ubiquitin ligase that protects cells from proteotoxic stress. The abilities of CHIP to interact with Hsp70 and function as a ubiquitin ligase place CHIP at a pivotal position in the protein quality control system, where its entrance into Hsp70-substrate complexes partitions nonnative proteins toward degradation. However, the manner by which Hsp70 substrates are selected for ubiquitination by CHIP is not well understood. We discovered that CHIP possesses an intrinsic chaperone activity that enables it to selectively recognize and bind nonnative proteins. Interestingly, the chaperone function of CHIP is temperature-sensitive and is dramatically enhanced by heat stress. The ability of CHIP to recognize nonnative protein structure may aid in selection of slow folding or misfolded polypeptides for ubiquitination.     

Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling.

BACKGROUND: Molecular chaperones recognize nonnative proteins and orchestrate cellular folding processes in conjunction with regulatory cofactors. However, not every attempt to fold a protein is successful, and misfolded proteins can be directed to the cellular degradation machinery for destruction. Molecular mechanisms underlying the cooperation of molecular chaperones with the degradation machinery remain largely enigmatic so far. RESULTS: By characterizing the chaperone cofactors BAG-1 and CHIP, we gained insight into the cooperation of the molecular chaperones Hsc70 and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin ligase in the ubiquitination of chaperone substrates such as the raf-1 protein kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, a ubiquitin domain protein previously shown to act as a coupling factor between Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone receptor in vivo. CONCLUSIONS: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control.     

Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl d-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.     

E3 ubiquitin-protein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity.     

Histone deacetylase inhibition decreases preference without affecting aversion for nicotine

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.     

A disease state mutation unfolds the parkin ubiquitin-like domain

E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.     

Parkin stabilizes microtubules through strong binding mediated by three independent domains

Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Our previous studies have demonstrated that parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. Here we show that the strong binding between parkin and tubulin, as well as that between parkin and microtubules, was mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuated colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules were not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates.     

Parkin interacts with LIM Kinase 1 and reduces its cofilin-phosphorylation activity via ubiquitination

Mutations in the PARKIN (PARK2) gene have been found in the majority of early-onset familial Parkinson's disease (PD) patients with autosomal recessive juvenile parkinsonism (ARJP). Parkin protein functions as an ubiquitin (E3) ligase that targets specific proteins for degradation in the 26S proteasome. Here, based on a mass spectrometry analysis of the human dopaminergic neuroblastoma-derived cell line SH-SY5Y that over-expresses parkin, we found that parkin may suppress cofilin phosphorylation. LIM Kinase 1 (LIMK1) is the upstream protein that phosphorylates cofilin, an actin depolymerizing protein. Thus, we postulated a possible connection between parkin and LIMK1. Our studies in other cell lines, using co-transfection assays, demonstrated that LIMK1 and parkin bind each other. LIMK1 also interacted with previously known parkin interactors Hsp70 and CHIP. Parkin enhanced LIMK1-ubiquitination in the human neuroblastoma-derived BE(2)-M17 cell line, but not in the human embryonic kidney-derived HEK293 cell line. In fact, parkin-over-expression reduced the level of LIMK1-induced phosphocofilin in the BE(2)-M17 cells but not in the HEK293 cells. Additionally, in simian kidney-derived COS-7 cells, parkin-over-expression reduced LIMK1-induced actin filament accumulation. LIMK1 in cultured cells regulates parkin reversibly: LIMK1 did not phosphorylate parkin but LIMK1 overexpression reduced parkin self-ubiquitination in vitro and in HEK293 cells. Furthermore, in the cells co-transfected with parkin and p38, LIMK1 significantly decreased p38-ubiquitination by parkin. These findings demonstrate a cell-type dependent functional interaction between parkin and LIMK1 and provide new evidence that links parkin and LIMK1 in the pathogenesis of familial PD.     

Parkin， Parkin 底物和帕金森病

Parkin 是隐性遗传性少年型帕金森病的致病基因 . 现认为 Parkin 行使泛素蛋白连接酶功能,参与蛋白质的泛素化过程 . 它的功能缺陷致使其底物蛋白质毒性积聚,从而介导多巴胺能神经元选择性死亡 . 越来越多的研究显示 Parkin 还具有神经保护作用,能对抗多种神经毒性刺激,并且可能参与路易体的形成过程,因此认为它在散发性帕金森病的致病过程中也可能起重要作用 .     

DNA damage induces nuclear translocation of parkin

Parkinson's disease (PD) is the second most common form of human degenerative disorder. Mutation of parkin is one of the most prevalent causes of autosomal recessive PD. Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here I show that parkin translocates into nucleus upon DNA damage. Nuclear translocation of parkin appears to be required to promote DNA repair. These findings suggest that DNA damage induces nuclear translocation of parkin leading to the PCNA interaction and possibly other nuclear proteins involved in DNA repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in parkinsonism.     

The co-chaperone CHIP is induced in various stresses and confers protection to cells

The C-terminus of Hsp70 interacting protein (CHIP) is being considered to be a cellular quality control E3 ubiquitin ligase because of its ability to degrade misfolded proteins in association with heat shock chaperones. The neuroprotective role of CHIP also has been implicated in several familial neurodegenerative diseases including polyglutamine diseases. However, the regulation of the expression of CHIP under different stress conditions and its protective role thereon is unknown. Here we have shown that the mRNA level of CHIP is significantly increased in the cells exposed to oxidative, endoplasmic reticulum and proteasomal stress. CHIP also protected from various stress-induced cell death. Finally, we have demonstrated upregulation of CHIP mRNA levels in the expanded polyglutamine protein expressing cells. Our result suggests that the upregulation of CHIP under various stress environments is an adaptive response of the cells to deal with the excess burden of misfolded protein.     

Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders

The study of neurodegenerative disorders has had a major impact on our understanding of more fundamental mechanisms underlying neurobiology. Breakthroughs in the genetics of Alzheimer's (AD) and Parkinson's diseases (PD) has resulted in new knowledge in the areas of axonal transport, energy metabolism, protein trafficking/clearance and synaptic physiology. The major neurodegenerative diseases have in common a regional or network pathology associated with abnormal protein accumulation(s) and various degrees of motor or cognitive decline. In AD, β-amyloids are deposited in extracellular diffuse and compacted plaques as well as intracellularly. There is a major contribution to the disease by the co-existence of an intraneuronal tauopathy. Additionally, PD-like Lewy Bodies (LBs) bearing aggregated α-synuclein is present in 40-60% of all AD cases, especially involving amygdala. Amyloid deposits can be degraded or cleared by several mechanisms, including immune-mediated and transcytosis across the blood-brain barrier. Another avenue for disposal involves the lysosome pathway via autophagy. Enzymatic pathways include insulin degradative enzyme and neprilysin. Finally, the co-operative actions of C-terminus Hsp70 interacting protein (CHIP) and Parkin, components of a multiprotein E3 ubiquitin ligase complex, may be a portal to proteasome-mediated degradation. Mutations in the Parkin gene are the most common genetic link to autosomal recessive Parkinson's disease. Parkin catalyzes the post-translational modification of proteins with polyubiquitin, targeting them to the 26S proteasome. Parkin reduces intracellular Aβ(1-42) peptide levels, counteracts its effects on cell death, and reverses its effect to inhibit the proteasome. Additionally, Parkin has intrinsic cytoprotective activity to promote proteasome function and defend against oxidative stress to mitochondria. Parkin and CHIP are also active in amyloid clearance and cytoprotection in vivo. Parkin has cross-functionality in additional neurodegenerative diseases, for instance, to eliminate polyglutamine-expanded proteins, reducing their aggregation and toxicity and reinstate proteasome function. The dual actions of CHIP (molecular co-chaperone and E3 ligase) and Parkin (as E3-ubiquitin ligase and anti-oxidant) may also play a role in suppressing inflammatory reactions in animal models of neurodegeneration. In this review, we focus on the significance of CHIP and Parkin as inducers of amyloid clearance, as cytoprotectants and in the suppression of reactive inflammation. A case is made for more effort to explore whether neurodegeneration associated with proteinopathies can be arrested at early stages by promoting their mutual action.     

E6-AP promotes misfolded polyglutamine proteins for proteasomal degradation and suppresses polyglutamine protein aggregation and toxicity

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases.     

Parkin mono-ubiquitinates Bcl-2 and regulates autophagy

Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. The mutations in the parkin gene can lead to a loss of function of parkin and cause autosomal recessive juvenile onset parkinsonism. Recently, parkin was reported to be involved in the regulation of mitophagy. Here, we identify the Bcl-2, an anti-apoptotic and autophagy inhibitory protein, as a substrate for parkin. Parkin directly binds to Bcl-2 via its C terminus and mediates the mono-ubiquitination of Bcl-2, which increases the steady-state levels of Bcl-2. Overexpression of parkin, but not its ligase-deficient forms, decreases autophagy marker LC3 conversion, whereas knockdown of parkin increases LC3 II levels. In HeLa cells, a parkin-deficient cell line, knockdown of parkin does not change LC3 conversion. Moreover, overexpression of parkin enhances the interactions between Bcl-2 and Beclin 1. Our results provide evidence that parkin mono-ubiquitinates Bcl-2 and regulates autophagy via Bcl-2.     

A product of the human gene adjacent to parkin is a component of Lewy bodies and suppresses Pael receptor-induced cell death

Parkin, a RING-type ubiquitin ligase, is the product of the gene responsible for autosomal recessive juvenile parkinsonism. A reverse strand gene located upstream of the parkin gene in the human genome has been identified. The gene product, termed Glup/PACRG, forms a large molecular chaperone complex containing heat shock proteins 70 and 90 and chaperonin components. Glup suppressed cell death induced by accumulation of unfolded Pael receptor (Pael-R), a substrate of Parkin. On the other hand, Glup facilitated the formation of inclusions consisting of Pael-R, molecular chaperones, protein degradation molecules, and Glup itself, when proteasome is inhibited. Glup knockdown attenuated the formation of Pael-R inclusions, which resulted in the promotion of cell death with extensive vacuolization. Moreover, Glup turned out to be a component of Lewy bodies in Parkinson's disease cases. These data suggest that Glup may play an important role in the formation of Lewy bodies and protection of dopaminergic neurons against Parkinson's disease.     

The Type II Hsp40 Sis1 Cooperates with Hsp70 and the E3 Ligase Ubr1 to Promote Degradation of Terminally Misfolded Cytosolic Protein

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.     

Inhibition of proteasomal activity causes inclusion formation in neuronal and non-neuronal cells overexpressing Parkin

Association between protein inclusions and neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, and polyglutamine disorders, has been widely documented. Although ubiquitin is conjugated to many of these aggregated proteins, the 26S proteasome does not efficiently degrade them. Mutations in the ubiquitin-protein ligase Parkin are associated with autosomal recessive juvenile Parkinsonism. Although Parkin-positive inclusions are not detected in brains of autosomal recessive juvenile Parkinsonism patients, Parkin is found in Lewy bodies in sporadic disease. This suggests that loss of Parkin ligase activity via mutation, or sequestration to Lewy bodies, is a contributory factor to sporadic disease onset. We now demonstrate that decreased proteasomal activity causes formation of large, noncytotoxic inclusions within the cytoplasm of both neuronal and nonneuronal cells overexpressing Parkin. This is not a general phenomenon as there is an absence of similar inclusions when HHARI, a structural homolog of Parkin, is overexpressed. The inclusions colocalize with ubiquitin and with proteasomes. Furthermore, Parkin inclusions colocalize with gamma-tubulin, acetylated alpha-tubulin, and cause redistribution of vimentin, suggesting aggresome-like properties. Our data imply that lower proteasomal activity, previously observed in brain tissue of Parkinson's disease patients, leads to Parkin accumulation and a concomitant reduction in ligase activity, thereby promoting Lewy body formation.