Hsp31, a member of the DJ-1 superfamily,is a multitasking stress responder with chaperone activity

Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses.     

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.     

MYO18B interacts with the proteasomal subunit Sug1 and is degraded by the ubiquitin-proteasome pathway

MYO18B is a class XVIIIB unconventional myosin encoded by a candidate tumor suppressor gene. To gain insights into the cellular function of this protein, we searched for MYO18B-interacting proteins by a yeast two-hybrid screen. Sug1, a 19S regulator subunit of the 26S proteasome, was identified as a binding partner of the C-terminal tail region of MYO18B. The association of MYO18B with Sug1 was further confirmed by GST pull-down, co-immunoprecipitation, and immunocytochemistry. Furthermore, proteasome dysfunction by a proteasome inhibitor or siRNA-mediated knock-down of Sug1 caused the up-regulation of MYO18B protein and MYO18B was polyubiquitinated in vivo. Collectively, these results suggested that MYO18B is a substrate for proteasomal degradation.     

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.     

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.     

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.     

Turnover of hepatitis B virus X protein is facilitated by Hdj1, a human Hsp40/DnaJ protein

Hepatitis B virus X (HBX) protein is required for the productive infection of hepatitis B virus (HBV) in vivo and implicated in the development of hepatocellular carcinoma. We have previously shown that hTid-1 and Hdj1, the human Hsp40/DnaJ chaperone proteins, bind the HBV core protein and inhibit viral replication in cell culture system. Here, we report evidences to suggest that HBX is the major target of Hdj1 in the inhibition of HBV replication. Expression of Hdj1 in cultured human hepatoma HepG2 cells facilitated degradation of HBX by the proteasome pathway, and thereby inhibited replication of the wild-type HBV as well as that of the HBX-deficient mutant virus rescued by HBX supplied in trans. Mutational analyses indicated that J domain of Hdj1 is required for the process. These results might provide a molecular basis for the antiviral effect of cellular chaperones.     

The Hsp70 chaperones of the Tritryps are characterized by unusual features and novel members

Proteins belonging to the Hsp70 class of molecular chaperones are highly conserved and ubiquitous, performing an essential role in the maintenance of cellular homeostasis in almost all known organisms. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are human parasites collectively known as the Tritryps. The Tritryps undergo extensive morphological changes during their life cycles, largely triggered by the marked differences between conditions in their insect vector and human host. Hsp70s are synthesised in response to these marked changes in environment and are proposed to be required for these parasites to successfully transition between differentiation stages while remaining viable and infective. While the Tritryps Hsp70 complement consists of homologues of all the major eukaryotic Hsp70s, there are a number of novel members, and some unique structural features. This review critically evaluates the current knowledge on the Tritryps Hsp70 proteins with an emphasis on T. brucei, and highlights some novel and previously unstudied aspects of these multifaceted molecular chaperones.     

The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms beta-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other.     

Differential regulation of NMDA receptor function by DJ‐1 and PINK1

Dysfunction of PTEN‐induced kinase 1 (PINK1) or DJ‐1 promotes neuronal death and is implicated in the pathogenesis of Parkinson’s disease, but the underlying mechanisms remain unclear. Given the roles of N‐methyl‐d‐ aspartate receptor (NMDAr)‐mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ‐1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr‐mediated whole‐cell currents by enhancing the function of NR2A‐containing NMDAr subtype (NR2ACNR). However, DJ‐1 decreased NMDAr‐mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B‐containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ‐1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ‐1‐dependent signal pathways DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ‐1, while promoted NMDA‐induced neuronal death through the overactivation of PTEN/NR2BCNR‐dependent cell death pathway, induced a neuroprotective effect to counteract DJ‐1 dysfunction‐mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival–promoting pathway. Thus, PINK1 acts with DJ‐1 in a common pathway to regulate NMDAr‐mediated neuronal death. This study suggests that the DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson’s disease.     

Thermal adaptation of the yeast mitochondrial Hsp70 system is regulated by the reversible unfolding of its nucleotide exchange factor

The Hsp70 protein switches during its functional cycle from an ADP-bound state with a high affinity for substrates to a low-affinity, ATP-bound state, with concomitant release of the client protein. The rate of the chaperone cycle is regulated by co-chaperones such as nucleotide exchange factors that significantly accelerate the ADP/ATP exchange. Mge1p, a mitochondrial matrix protein with homology to bacterial GrpE, serves as the nucleotide exchange factor of mitochondrial Hsp70. Here, we analyze the influence of temperature on the structure and functional properties of Mge1p from the yeast Saccharomyces cerevisiae. Mge1p is a dimer in solution that undergoes a reversible thermal transition at heat-shock temperatures, i.e. above 37 degrees C, that involves protein unfolding and dimer dissociation. The thermally denatured protein is unable to interact stably with mitochondrial Hsp70, and therefore is unable to regulate its ATPase and chaperone cycle. Crosslinking of wild-type mitochondria reveals that Mge1p undergoes the same dimer to monomer temperature-dependent shift, and that the nucleotide exchange factor does not associate with its Hsp70 partner at stress temperatures (i.e. > or =45 degrees C). Once the stress conditions disappear, Mge1p refolds and recovers both structure and functional properties. Therefore, Mge1p can act as a thermosensor for the mitochondrial Hsp70 system, regulating the nucleotide exchange rates under heat shock, as has been described for two bacterial GrpE proteins. The thermosensor activity is conserved in the GrpE-like nucleotide exchange factors although, as discussed here, it is achieved through a different structural mechanism.     

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.     

Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1

Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin-proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N-end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.     

The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N -glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae , we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis -prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2 , only HMG2 overexpression was able to restore growth of the yta7 Δ cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.     

Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway

Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.     

HspBP1, a homologue of the yeast Fes1 and Sls1 proteins,is an Hsc70 nucleotide exchange factor

The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s, Ssa1p and BiP, respectively. We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70. In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro. Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity.     

The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR^mt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.