The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein

DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.     

Dissection of the dimerization modes in the DJ-1 superfamily

The DJ-1 superfamily (DJ-1/ThiJ/PfpI superfamily) is distributed across all three kingdoms of life. These proteins are involved in a highly diverse range of cellular functions, including chaperone and protease activity. DJ-1 proteins usually form dimers or hexamers in vivo and show at least four different binding orientations via distinct interface patches. Abnormal oligomerization of human DJ-1 is related to neurodegenerative disorders including Parkinson’s disease, suggesting important functional roles of quaternary structures. However, the quaternary structures of the DJ-1 superfamily have not been extensively studied. Here, we focus on the diverse oligomerization modes among the DJ-1 superfamily proteins and investigate the functional roles of quaternary structures both computationally and experimentally. The oligomerization modes are classified into 4 types (DJ-1, YhbO, Hsp, and YDR types) depending on the distinct interface patches (I-IV) upon dimerization. A unique, rotated interface via patch I is reported, which may potentially be related to higher order oligomerization. In general, the groups based on sequence similarity are consistent with the quaternary structural classes, but their biochemical functions cannot be directly inferred using sequence information alone. The observed phyletic pattern suggests the dynamic nature of quaternary structures in the course of evolution. The amino acid residues at the interfaces tend to show lower mutation rates than those of non-interfacial surfaces.     

Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress

DJ-1 is a novel oncogene and causative gene for familial form of the Parkinson's disease (PD). DJ-1 has been shown to play a role in anti-oxidative stress by eliminating reactive oxygen species (ROS). The onset of PD is thought to be caused by oxidative stress and mitochondrial injury, which leads to protein aggregation that results in neuronal cell death. However, the mechanism by which DJ-1 triggers the onset of PD is still not clear. In this study, we analyzed association and localization of DJ-1 and its mutants with various chaperones. The results showed that DJ-1 and its mutants were associated with Hsp70, CHIP and mtHsp70/Grp75, a mitochondria-resident Hsp70, and that L166P and M26I mutants found in PD patients were strongly associated with Hsp70 and CHIP compared to wild-type and other DJ-1 mutants. DJ-1 and its mutants were colocalized with Hsp70 and CHIP in cells. Furthermore, association and colocalization of wildtype DJ-1 with mtHsp70 in mitochondria were found to be enhanced by treatment of cells with H₂O₂. These results suggest that translocation of DJ-1 to mitochondria after oxidative stress is carried out in association with chaperones.     

Zinc-mediated Reversible Multimerization of Hsp31 Enhances the Activity of Holding Chaperone

Hsp31 protein, belonging to the DJ-1/ThiJ/PfpI superfamily, increases the survival of Escherichia coli under various stresses. While it was reported as a holding chaperone, Hsp31 was also shown to exhibit the glyoxalase III activity in subsequent study. Here, we describe our finding that Hsp31 undergoes a Zn^+ 2-mediated multimerization (HMW_Zinc), resulting in an enhanced chaperone activity. Furthermore, it was shown that the formation of HMW_Zinc is reversible such that the oligomer dissociates into the native dimer by EDTA incubation. We attempted to determine the structural change involving the transition between the native dimer and HMW_Zinc by adding Ni^+ 2, which is Zn^+ 2-mimetic, producing a potential intermediate structure. An analysis of this intermediate revealed a structure with hydrophobic interior exposed, due to an unfolding of the N-terminal loop and the C-terminal β-to-α region. A treatment with hydrogen peroxide accelerated HMW_Zinc formation, so that the Hsp31^C185E mutant rendered the formation of HMW_Zinc even at 45 °C. However, the presence of Zn^+ 2 in the catalytic site antagonizes the oxidation of C185, implying a negative role. Our results suggest an unprecedented mechanism of the enhancing chaperone activity by Hsp31, in which the reversible formation of HMW_Zinc occurs in the presence of heat and Zn^+ 2 ion.     

The molecular chaperone Hsp90 is required for high osmotic stress response in Saccharomyces cerevisiae

Exposure of Saccharomyces cerevisiae to high osmotic stress evokes a number of adaptive changes that are necessary for its survival. These adaptive responses are mediated via multiple mitogen-activated protein kinase pathways, of which the high-osmolarity glycerol (HOG) pathway has been studied most extensively. Yeast strains that bear the hsp82T22I or hsp82G81S mutant alleles are osmosensitive. Interestingly, the osmosensitive phenotype is not due to inappropriate functioning of the HOG pathway, as Hog1p phosphorylation and downstream responses including glycerol accumulation are not affected. Rather, the hsp82 mutants display features that are characteristic for cell-wall mutants, i.e. resistance to Zymolyase and sensitivity to Calcofluor White. The osmosensitivity of the hsp82T22I or hsp82G81S strains is suppressed by over-expression of the Hsp90 co-chaperone Cdc37p but not by other co-chaperones. Hsp90 is shown to be required for proper adaptation to high osmolarity via a novel signal transduction pathway that operates parallel to the HOG pathway and requires Cdc37p.     

DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein

DJ-1 is a ubiquitously expressed protein involved in various cellular processes including cell proliferation, RNA-binding, and oxidative stress. Mutations that result in loss of DJ-1 function lead to early onset parkinsonism in humans, and DJ-1 protein is present in pathological lesions of several tauopathies and synucleinopathies. In order to further investigate the role of DJ-1 in human neurodegenerative disease, we have generated novel polyclonal and monoclonal antibodies to human DJ-1 protein. We have characterized these antibodies and confirmed the pathological co-localization of DJ-1 with other neurodegenerative disease-associated proteins, as well as the decrease in DJ-1 solubility in disease tissue. In addition, we report the presence of DJ-1 in a large molecular complex (> 2000 kDa), and provide evidence for an interaction between endogenous DJ-1 and alpha-synuclein in normal and diseased tissue. These findings provide new avenues towards the study of DJ-1 function and how loss of its activity may lead to parkinsonism. Furthermore, our results provide further evidence for the interplay between neurodegenerative disease-associated proteins.     

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.     

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.     

The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle

The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.     

Free radicals impair the anti-oxidative stress activity of DJ-1 through the formation of SDS-resistant dimer

DJ-1 is a causative gene for familial Parkinson’s disease (PD). Loss-of-function of DJ-1 protein is suggested to contribute to the onset of PD, but the causes of DJ-1 dysfunction remain insufficiently elucidated. In this study, we found that the SDS-resistant irreversible dimer of DJ-1 protein was formed in human dopaminergic neuroblastoma SH-SY5Y cells when the cells were exposed to massive superoxide inducers such as paraquat and diquat. The dimer was also formed in vitro by superoxide in PQ redox cycling system and hydroxyl radical produced in Fenton reaction. We, thus, found a novel phenomenon that free radicals directly affect DJ-1 to form SDS-resistant dimers. Moreover, the formation of the SDS-resistant dimer impaired anti-oxidative stress activity of DJ-1 both in cell viability assay and H₂O₂-elimination assay in vitro. Similar SDS-resistant dimers were steadily formed with several mutants of DJ-1 found in familial PD patients. These findings suggest that DJ-1 is impaired due to the formation of SDS-resistant dimer when the protein is directly attacked by free radicals yielded by external and internal stresses and that the DJ-1 impairment is one of the causes of sporadic PD.     

A Glutathione-independent Glyoxalase of the DJ-1 Superfamily Plays an Important Role in Managing Metabolically Generated Methylglyoxal in Candida albicans

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys¹³⁶-His¹³⁷-Glu¹⁶⁸. Purified Glx3p has an in vitro methylglyoxalase activity (K_m = 5.5 mm and k_cat = 7.8 s⁻¹) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.     

The atomic resolution crystal structure of the YajL (ThiJ) protein from Escherichia coli: a close prokaryotic homologue of the Parkinsonism-associated protein DJ-1

The Escherichia coli protein YajL (ThiJ) is a member of the DJ-1 superfamily with close homologues in many prokaryotes. YajL also shares 40% sequence identity with human DJ-1, an oncogene and neuroprotective protein whose loss-of-function mutants are associated with certain types of familial, autosomal recessive Parkinsonism. We report the 1.1 angstroms resolution crystal structure of YajL in a crystal form with two molecules in the asymmetric unit. The structure of YajL is remarkably similar to that of human DJ-1 (0.9 angstroms C(alpha) RMSD) and both proteins adopt the same dimeric structure. The conserved cysteine residue located in the "nucleophile elbow" is oxidized to either cysteine sulfenic or sulfinic acid in the two molecules in the asymmetric unit, and a mechanism for this oxidation is proposed that may be valid for other proteins in the DJ-1 superfamily as well. Rosenfield difference matrix analysis of the refined anisotropic displacement parameters in the YajL structure reveals significant differences in the intramolecular flexibility of the two non-crystallographic symmetry-related molecules in the asymmetric unit. Lastly, a comparison of the crystal structures of the four different E.coli members of the DJ-1 superfamily indicates that the variable oligomerization in this superfamily is due to a combination of protein-specific insertions into the core fold that form specific interfaces while occluding others plus optimization of residues in the structurally invariant regions of the core fold that facilitate protein-protein interactions.     

Interaction of Hsp70 with p49/STRAP, a serum response factor binding protein

Members of the Hsp70 protein family must work with other co-chaperones to exert their function. Herein, we identified a new Hsp70 co-chaperone, p49/STRAP, previously shown to interact with serum response factor. We demonstrated that a fraction of p49/STRAP was cytosolic, and that it interacted with the β-sandwich domain of Hsp70. Although p49/STRAP had little effect on the intrinsic ATPase activity of Hsp70, it reduced the ATP-hydrolytic activity of Hsp70 stimulated by Hsp40, and inhibited the refolding activity of the Hsp70/Hsp40 system. Thus, p49/STRAP can be considered a bona fide co-chaperone of Hsp70.     

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H₂O₂, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.     

Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition

Mutations in DJ-1 gene have been linked to autosomal recessive early onset parkinsonism (AR-EOP). Although the mechanism of neuronal cell death due to DJ-1 mutation has not been fully elucidated, loss of DJ-1 function was considered to cause the phenotype. Here, we demonstrated that the down regulation of endogenous DJ-1 of the neuronal cell line by siRNA enhanced the cell death which was induced by oxidative stress, ER stress, and proteasome inhibition, but not by pro-apoptotic stimulus. The cell death with hydrogen peroxide was dramatically rescued by over-expression of wild-type DJ-1, but not by that of L166P mutant DJ-1. Furthermore, DJ-1 rescued the cell death caused by over-expression of Pael receptor, which was a substrate of Parkin, another gene product for autosomal recessive juvenile parkinsonism. These results suggest that loss of protective activity of DJ-1 from neuro-toxicity induced by these stresses contributes to neuronal cell death in AR-EOP with mutant DJ-1.     

DJ-1 enhances cell survival through the binding of Cezanne, a negative regulator of NF-kappaB

Heightened DJ-1 (Park7) expression is associated with a reduction in chemotherapeutic-induced cell death and poor prognosis in several cancers, whereas the loss of DJ-1 function is found in a subgroup of Parkinson disease associated with neuronal death. This study describes a novel pathway by which DJ-1 modulates cell survival. Mass spectrometry shows that DJ-1 interacts with BBS1, CLCF1, MTREF, and Cezanne/OTUD7B/Za20d1. Among these, Cezanne is a known deubiquitination enzyme that inhibits NF-κB activity. DJ-1/Cezanne interaction is confirmed by co-immunoprecipitation of overexpressed and endogenous proteins, maps to the amino-terminal 70 residues of DJ-1, and leads to the inhibition of the deubiquitinating activity of Cezanne. Microarray profiling of shRNA-transduced cells shows that DJ-1 and Cezanne regulate IL-8 and ICAM-1 expression in opposing directions. Similarly, DJ-1 enhances NF-κB nuclear translocation and cell survival, whereas Cezanne reduces these outcomes. Analysis of mouse Park7(-/-) primary cells confirms the regulation of ICAM-1 by DJ-1 and Cezanne. As NF-κB is important in cellular survival and transformation, IL-8 functions as an angiogenic factor and pro-survival signal, and ICAM-1 has been implicated in tumor progression, invasion, and metastasis; these data provide an additional modality by which DJ-1 controls cell survival and possibly tumor progression via interaction with Cezanne.     

The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1

Melusin is a mammalian muscle specific CHORD containing protein capable of activating signal transduction pathways leading to cardiomyocytes hypertrophy in response to mechanical stress. To define melusin function we searched for molecular partners possibly involved in melusin dependent signal transduction. Here we show that melusin and heat shock proteins are co-regulated. Moreover, melusin directly binds to Hsp90, a ubiquitous chaperone involved in regulating several signaling pathways. In addition, melusin interacts with Sgt1, an Hsp90 binding molecule. Melusin does not behave as an Hsp90 substrate but rather as a chaperone capable to protect citrate synthase from heat induced aggregation. These results describe melusin as a new component of the Hsp90 chaperone machinery.     

Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane

Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins. The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif. Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro. Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70. Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.