Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes

Although the ubiquitin-proteasome system and the molecular chaperones are implicated to play an important role in pathogenesis of familial amyotrophic lateral sclerosis (FALS) caused by mutations in Cu/Zn-superoxide dismutase (SOD1), the mechanism underlying the causes of this fatal disease is still poorly understood. Here we found that co-chaperone CHIP (carboxyl terminus of Hsc70-interacting protein), together with molecular chaperones Hsc70/Hsp70 and Hsp90, associates with FALS-linked mutant SOD1 proteins in cultured human cells. S5a subunit of 26S proteasomes, which recognizes polyubiquitylated proteins, also interacts with mutant SOD1 proteins. Over-expression of CHIP leads to the reduction in cellular levels of mutant SOD1 as well as the suppression of cytotoxicity induced by mutant SOD1. Unusually, rather than increasing the level of poly-ubiquitylated SOD1, over-expressed CHIP alters the ubiquitylation pattern of mutant SOD1 proteins. Both down-regulation and ubiquitylation of mutant SOD1 are greatly reduced by a mutant CHIP protein lacking U-box domain. Taken together, these results suggest that co-chaperone CHIP, possibly with another E3 ligase(s), modulates the ubiquitylation of mutant SOD1 and renders them more susceptible for proteasomal degradation.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

The molecular chaperone heat shock protein 90 (Hsp90) and the co-chaperone/ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) control the turnover of client proteins. How this system decides to stabilize or degrade the client proteins under particular physiological or pathological conditions is unclear. We report here a novel client protein, the SUMO2/3 protease SENP3, that is sophisticatedly regulated by CHIP and Hsp90. SENP3 is maintained at a low basal level under non-stress condition due to Hsp90-independent CHIP-mediated ubiquitination. Upon mild oxidative stress, SENP3 undergoes thiol modification, which recruits Hsp90. Hsp90/SENP3 association protects SENP3 from CHIP-mediated ubiquitination and subsequent degradation, but this effect of Hsp90 requires the presence of CHIP. Our data demonstrate for the first time that CHIP and Hsp90 interplay with a client alternately under non-stress and stress conditions, and the choice between stabilization and degradation is made by the redox state of the client. In addition, enhanced SENP3/Hsp90 association is found in cancer. These findings provide new mechanistic insight into how cells regulate the SUMO protease in response to oxidative stress.     

C-terminus of heat shock protein 70-interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1-dependent apoptosis

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress.     

Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging

We previously reported the disruption of caveolae/rafts, dysfunction of Golgi tethers, N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptor proteins (SNAREs), and SNAPs, and inhibition of anterograde trafficking in endothelial cells in culture and rat lung exposed to monocrotaline pyrrole (MCTP) as a prelude to the development of pulmonary hypertension. We have now investigated 1) whether this trafficking block affects subcellular localization and function of endothelial nitric oxide (NO) synthase (eNOS) and 2) whether Golgi blockade and eNOS sequestration are observed after hypoxia and senescence. Immunofluorescence data revealed that MCTP-induced "megalocytosis" of pulmonary arterial endothelial cells (PAEC) was accompanied by a loss of eNOS from the plasma membrane, with increased accumulation in the cytoplasm. This cytoplasmic eNOS was sequestered in heterogeneous compartments and partially colocalized with Golgi and endoplasmic reticulum (ER) markers, caveolin-1, NOSTRIN, and ER Tracker, but not Lyso Tracker. Hypoxia and senescence also produced enlarged PAEC, with dysfunctional Golgi and loss of eNOS from the plasma membrane, with sequestration in the cytoplasm. Live-cell imaging of caveolar and cytoplasmic NO with 4,5-diaminofluorescein diacetate (DAF-2DA) as probe showed a marked loss of caveolar NO after MCTP, hypoxia, and senescence. Although ionomycin stimulated DAF-2DA fluorescence in control PAEC, this ionophore decreased DAF-2DA fluorescence in MCTP-treated and senescent PAEC, suggesting localization of eNOS in an aberrant cytoplasmic compartment that was readily discharged by Ca(2+)-induced exocytosis. Thus monocrotaline, hypoxia, and senescence produce a Golgi blockade in PAEC, leading to sequestration of eNOS away from its functional caveolar location and providing a mechanism for the often-reported reduction in pulmonary arterial NO levels in experimental pulmonary hypertension, despite sustained eNOS protein levels.     

Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70.BAG-1.CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.     

Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes

Diabetes mellitus (DM) and high glucose (HG) are known to reduce the bioavailability of nitric oxide (NO) by modulating endothelial nitric-oxide synthase (eNOS) activity. eNOS is regulated by several mechanisms including its interaction with heat shock protein (Hsp) 90. We previously discovered that DM in vivo and HG in vitro induced the translocation of Hsp90alpha to the outside of aortic endothelial cells. In this report we tested the hypothesis that translocation of Hsp90alpha is responsible for the decline in NO production observed in HG-treated cells. We found that HG increased phosphorylation of Hsp90alpha in a cAMP-dependent protein kinase A-dependent manner, and that this event was required for translocation of Hsp90alpha in porcine aortic endothelial cells. Furthermore, preventing translocation of Hsp90alpha protected from the HG-induced decline in eNOS.Hsp90alpha complex and NO production. Notably, DM increased phosphorylation of Hsp90alpha and reduced its association with eNOS in the aortic endothelium of diabetic rats. These studies suggest that translocation of Hsp90alpha is a novel mechanism by which HG and DM impair eNOS activity and thereby reduce NO production.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture

We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.     

Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

Protein quality control: U-box-containing E3 ubiquitin ligases join the fold

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.     

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.     

The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase

The ubiquitin–proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C−Cullin-5−SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70–interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime.     

Plasma membrane-associated endothelial nitric oxide synthase and activity in aging rat aortic vascular endothelia markedly decline with age

The mechanisms leading to the age-related loss of endothelial nitric oxide (NO) and NO-dependent vasodilation remain largely unknown. Freshly isolated endothelium from young (6 months) and old (36 months) F344xBrN rats were analyzed for endothelial nitric oxide synthase (eNOS) protein, its subcellular distribution, and association with regulatory proteins. Results show that both vessel ring vasoreactivity and A23187-induced eNOS activity in isolated endothelial cells significantly (p < or = 0.05) declined with age. Levels of cGMP, a reliable marker for NO bioactivity also declined significantly (p < or = 0.01). However, no change in overall eNOS protein was evident. Subcellular fractionation studies revealed an age-related loss in active, plasma membrane-bound eNOS relative to eNOS in the Golgi/cytosol of the endothelium. Plasma membrane-associated eNOS in aged endothelium was also less complexed with the activating proteins Hsp90 and Akt and more associated with to caveolin-1, which inhibits eNOS activity. These results suggest that age-dependent loss of NO may be partly caused by differences in eNOS subcellular distribution and its association with inhibitory proteins.     

Src-mediated phosphorylation of Hsp90 in response to vascular endothelial growth factor (VEGF) is required for VEGF receptor-2 signaling to endothelial NO synthase

Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated by an Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heat-shock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90beta. Tyrosine phosphorylation of Hsp90beta in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells.     

Regulation of the cytoplasmic quality control protein degradation pathway by BAG2

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.     

Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase

The balance of nitric oxide (.NO) and superoxide anion (O(2)) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates.NO. As eNOS is capable of generating both.NO and O(2), we hypothesized that Hsp90 might also mediate eNOS-dependent O(2) production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 microg/ml; 17.8 microm) and then stimulated with the calcium ionophore, (5 microm). GA significantly decreased -stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased -stimulated eNOS-dependent O(2) production (p < 0.001, n = 8). increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent.NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O(2) production.     

CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C-terminus of Hsc70-interacting protein (CHIP) with a U-box domain is an E3 ubiquitin-ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U-box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C-terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self-ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality-control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.     

Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.     

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.