Parkin facilitates the elimination of expanded polyglutamine proteins and leads to preservation of proteasome function

Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated alpha-synuclein, Pael-R, and synphilin-1. We now show that parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Using a cellular proteasome indicator system based on a destabilized form of green fluorescent protein, we demonstrate that parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome, which may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. We speculate that parkin may function to relieve endoplasmic reticulum stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's disease.     

CHIP is associated with Parkin,a gene responsible for familial Parkinson's disease,and enhances its ubiquitin ligase activity

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.     

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.     

Compensatory function of transthyretin in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of age-related primary neurodegenerative diseases characterized by progressive memory loss, aphasia, and intellectual and mental breakdown. Pathogenesis of AD is based on the early synaptic dysfunction following neurodegeneration and neuronal death. According to modern concepts, the development of neuropathological processes is due to progressively deposited intermediates of amyloid fibrils that represent oligomers consisting of short peptide named amyloid beta protein (Abeta). In this context, it is reasonable to propose that one of the compensatory mechanisms of AD might be inhibition of Abeta oligomerization by sequestration or clearance of Abeta. Experiments with transgenic animals and epidemiological studies demonstrate that major protein of cerebrospinal fluid, transthyretin, is a natural neuroprotector that inhibits Abeta amyloid formation and restore cognitive functions. The study of Abeta-transthyretin complexes allowed to create peptides that are mimetics of transthyretin. These mimetics inhibit amyloid formation in vitro and, therefore, could be used in therapeutic treatment of AD.     

Ubiquitinylation of α-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is regulated by Bcl-2-associated athanogene 5 (BAG5)

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization.     

The downward spiral of tau and autolysosomes

A growing body of research has connected autophagy to neurodegenerative diseases such as Alzheimer disease (AD). In autopsied AD brain, large multivesicular bodies accumulate in neurons. Knockout mice deficient for key autophagy genes demonstrate age-dependent neurodegeneration. Most neurodegenerative diseases are characterized by accumulation of insoluble protein species; the type of protein and the location of aggregates within the nervous system help to define the type of disorder. It has been hypothesized that the inability to degrade such aggregates is a major causative factor in neuronal dysfunction and eventual neuronal death. As neurons are postmitotic and thus cannot regenerate themselves, mechanisms of protein clearance have received much attention in the field. The function of the ubiquitin-proteasome system (UPS) is impaired in models of neurodegeneration, and overexpression of chaperone proteins, such as those in the HSP70 family, leads to beneficial effects in many models of proteinopathies. Recently, studies of the effects of autophagy as a clearance mechanism have uncovered compelling evidence that inducing autophagy can alleviate many pathogenic and behavioral symptoms in animal and cellular models of neurodegeneration.     

The downward spiral of tau and autolysosomes: A new hypothesis in neurodegeneration

A growing body of research has connected autophagy to neurodegenerative diseases such as Alzheimer disease (AD). In autopsied AD brain, large multivesicular bodies accumulate in neurons. Knockout mice deficient for key autophagy genes demonstrate age-dependent neurodegeneration. Most neurodegenerative diseases are characterized by accumulation of insoluble protein species; the type of protein and the location of aggregates within the nervous system help to define the type of disorder. It has been hypothesized that the inability to degrade such aggregates is a major causative factor in neuronal dysfunction and eventual neuronal death. As neurons are postmitotic and thus cannot regenerate themselves, mechanisms of protein clearance have received much attention in the field. The function of the ubiquitin-proteasome system (UPS) is impaired in models of neurodegeneration, and overexpression of chaperone proteins, such as those in the HSP70 family, leads to beneficial effects in many models of proteinopathies. Recently, studies of the effects of autophagy as a clearance mechanism have uncovered compelling evidence that inducing autophagy can alleviate many pathogenic and behavioral symptoms in animal and cellular models of neurodegeneration.     

Misfolded Proteins Recognition Strategies of E3 Ubiquitin Ligases and Neurodegenerative Diseases

Impairment in the clearance of misfolded proteins by functional proteins leads to various late-onset neurodegenerative diseases. Cell applies a strict quality control mechanism against malfunctioned proteins which may generate cellular proteoxicity. Under proteotoxic insults, cells immediately adopt two major approaches to either refold the misfolded proteinaceous species or degrade the unmanageable candidates. However, the main cellular proteostasis quality control mechanism is not clear. It is therefore important to understand the events and cellular pathways, which are implicated in the clearance of recalcitrant proteins. Ubiquitin proteasome system manages intracellular protein degradation. In this process, E3 ubiquitin ligase enzyme provides specificity for recognition of client proteins. In this review, we summarize various molecular approaches governed by E3 ubiquitin ligases in the degradation of aberrant proteins. A clear understanding of E3 ubiquitin ligases can offer a well tractable therapeutic approach against neurodegenerative diseases.     

Alternative effects of the ubiquitin-proteasome pathway on glucocorticoid receptor down-regulation and transactivation are mediated by CHIP, an E3 ligase

The ubiquitin/proteasome-dependent protein degradation pathway (UPP) is responsible for the accelerated down-regulation of glucocorticoid receptor (GR) levels in cells subjected to chronic glucocorticoid exposure. Whereas hormone-dependent down-regulation of GR operates in most cells, the receptor is not down-regulated after long-term glucocorticoid treatment of either cultured embryonic hippocampal neurons or the HT22 hippocampal cell line. In this report, we show that stable overexpression of the carboxy terminus of heat shock protein 70-interacting protein (CHIP) E3 ligase can restore hormone-dependent down-regulation of GR in HT22 cells. Proteasome inhibitor studies establish that ubiquitylated GR can be efficiently engaged with the proteasome upon CHIP overexpression, unlike the case in parental HT22 cells. In addition to its impact on GR down-regulation, CHIP overexpression alters the coupling between the UPP and GR transactivation. Unlike other steroid receptors whose transactivation properties are typically reduced upon proteasome inhibition, GR transactivation in HT22 cells and other cell lines is enhanced upon proteasome inhibition. However, in HT22 cells overexpressing CHIP, proteasome inhibition leads to a reduction in GR transactivation activity. Thus, the divergent response of a single transactivator (i.e. GR) to the UPP can be dictated by CHIP, an E3 ligase that also functions as a proteasome-targeting factor.     

Drosophila melanogaster Parkin ubiquitinates peanut and septin1 as an E3 ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.     

Autoregulation of Parkin activity through its ubiquitin-like domain

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.     

Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons

One hypothesis for the etiology of Parkinson's disease (PD) is that subsets of neurons are vulnerable to a failure in proteasome-mediated protein turnover. Here we show that overexpression of mutant alpha-synuclein increases sensitivity to proteasome inhibitors by decreasing proteasome function. Overexpression of parkin decreases sensitivity to proteasome inhibitors in a manner dependent on parkin's ubiquitin-protein E3 ligase activity, and antisense knockdown of parkin increases sensitivity to proteasome inhibitors. Mutant alpha-synuclein also causes selective toxicity to catecholaminergic neurons in primary midbrain cultures, an effect that can be mimicked by the application of proteasome inhibitors. Parkin is capable of rescuing the toxic effects of mutant alpha-synuclein or proteasome inhibition in these cells. Therefore, parkin and alpha-synuclein are linked by common effects on a pathway associated with selective cell death in catecholaminergic neurons.     

BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP

Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.     

CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation

Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chaperones such as Hsc/Hsp70 are assisted by cofactors that modulate the folding machinery in a positive or negative manner. CHIP (carboxyl terminus of Hsc70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. Using an in vitro ubiquitylation assay with recombinant proteins, we demonstrate that CHIP possesses intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiquitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70, primarily with short, noncanonical multiubiquitin chains but has no appreciable effect on steady-state levels or half-life of this protein. This effect may have heretofore unanticipated consequences with regard to the chaperoning activities of Hsc70 or its ability to deliver substrates to the proteasome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E3s.     

Parkin Somatic Mutations Link Melanoma and Parkinson's Disease

Epidemiological studies suggest a direct link between melanoma and Parkinson's disease(PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28 K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.     

In search of genes involved in neurodegenerative disorders

Dissecting the genetics of Alzheimer's disease (AD) and Parkinson's disease (PD) has contributed significantly to our understanding of the pathogenesis of neurodegeneration in these two complex disorders. For AD, three highly penetrant genes (amyloid precursor protein (APP, PSEN1 and PSEN2) and one susceptibility gene (APOE) have been identified. For PD, seven genes (SNCA, Parkin, UCHL1, NR4A2, DJ1, PINK1 and LRRK2) have been found. These genes explain only a small proportion of AD and PD patients and are mostly associated with an early onset presentation of the disease. APOE remains the only common gene, which increases the risk of both rare early and late onset AD. The ongoing challenge is to unravel the genetics of the most frequent forms of these complex disorders. In the present paper, we briefly review the state of the art in the genetics of AD and PD. We also discuss the prospects of finding new genes associated with common forms of these diseases in light of two hypotheses concerning the genetic variation of complex diseases: common disease/common variants and common disease/rare variants.     

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.     

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.     

U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.