Hsp90 and co‐chaperones twist the functions of diverse client proteins

Hsp90 molecular chaperones are required for the stability and activity of a diverse range of client proteins that have critical roles in signal transduction, cellular trafficking, chromatin remodeling, cell growth, differentiation, and reproduction. Mammalian cells contain three types of Hsp90s: cytosolic Hsp90, mitochondrial Trap‐1, and Grp94 of the endoplasmic reticulum. Each of the Hsp90s, as well as the bacterial homolog, HtpG, hydrolyze ATP and undergo similar conformational changes. Unlike the other forms of Hsp90, cytosolic Hsp90 function is dependent on a battery of co‐chaperone proteins that regulate the ATPase activity of Hsp90 or direct Hsp90 to interact with specific client proteins. This review will summarize what is known about Hsp90's ability to mediate the folding and activation of diverse client proteins that contribute to human diseases, such as cancer and fungal and viral infections. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 211–217, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structure and function of the molecular chaperone Hsp104 from yeast

The molecular chaperone Hsp104 plays a central role in the clearance of aggregates after heat shock and the propagation of yeast prions. Hsp104's disaggregation activity and prion propagation have been linked to its ability to resolubilize or remodel protein aggregates. However, Hsp104 has also the capacity to catalyze protein aggregation of some substrates at specific conditions. Hence, it is a molecular chaperone with two opposing activities with respect to protein aggregation. In yeast models of Huntington's disease, Hsp104 is required for the aggregation and toxicity of polyglutamine (polyQ), but the expression of Hsp104 in cellular and animal models of Huntington's and Parkinson's disease protects against polyQ and α‐synuclein toxicity. Therefore, elucidating the molecular determinants and mechanisms underlying the ability of Hsp104 to switch between these two activities is of critical importance for understanding its function and could provide insight into novel strategies aimed at preventing or reversing the formation of toxic protein aggregation in systemic and neurodegenerative protein misfolding diseases. Here, we present an overview of the current molecular models and hypotheses that have been proposed to explain the role of Hsp104 in modulating protein aggregation and prion propagation. The experimental approaches and the evidences presented so far in relation to these models are examined. Our primary objective is to offer a critical review that will inspire the use of novel techniques and the design of new experiments to proceed towards a qualitative and quantitative understanding of the molecular mechanisms underlying the multifunctional properties of Hsp104 in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 93:252–276, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Interplay between protein homeostasis networks in protein aggregation and proteotoxicity

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self‐association of disease proteins and determine whether they elicit a toxic or benign outcome. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 229–236, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Association of heat-shock proteins in various neurodegenerative disorders: is it a master key to open the therapeutic door?

A number of acute and chronic neurodegenerative disorders are caused due to misfolding and aggregation of many intra- and extracellular proteins. Protein misfolding and aggregation processes in cells are strongly regulated by cellular molecular chaperones known as heat-shock proteins (Hsps) that include Hsp60, Hsp70, Hsp40, and Hsp90. Recent studies have shown the evidences that Hsps are colocalized in protein aggregates in Alzheimer’s disease (AD), Parkinson’s disease (PD), Polyglutamine disease (PGD), Prion disease, and other neurodegenerative disorders. This fact indicates that Hsps might have attempted to prevent aggregate formation in cells and thus to suppress disease conditions. Experimental findings have already established in many cases that selective overexpression of Hsps like Hsp70 and Hsp40 prevented the disease progression in various animal models and cellular models. However, recently, various Hsp modulators like geldanamycin, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, and celastrol have shown to up-regulate the expression level of Hsp70 and Hsp40, which in turn triggers the solubilization of diseased protein aggregates. Hsps are, therefore, if appropriately selected, an attractive choice for therapeutic targeting in various kinds of neurodegeneration and hence are expected to have strong potential as therapeutic agents in suppressing or curing AD, PD, PGD, and other devastative neurodegenerative disorders. In the present review, we report the experimental findings that describe the implication of Hsps in the development of neurodegeneration and explore the possibility of how Hsps can be used directly or as a target by other agents to prevent various neurodegeneration through preventing aggregation process and thus reducing the toxicity of the oligomers based on the previous reports.     

Insight into the stability of cross‐β amyloid fibril from molecular dynamics simulation

Amyloid fibrils are considered to play causal roles in the pathogenesis of amyloid‐related degenerative diseases such as Alzheimer's disease, type II diabetes mellitus, the transmissible spongiform encephalopathies, and prion disease. The mechanism of fibril formation is still hotly debated and remains an important open question. In this study, we utilized molecular dynamics (MD) simulation to analyze the stability of hexamer for eight class peptides. The MD results suggest that VEALYL and MVGGVV‐1 are the most stable ones, then SNQNNY, followed by LYQLEN, MVGGVV‐2, VQIVYK, SSTSAA, and GGVVIA. The statistics result indicates that hydrophobic residues play a key role in stabilizing the zipper interface. Single point and two linkage mutants of MVGGVV‐1 confirmed that both Met1 and Val2 are key hydrophobic residues. This is consistent with the statistics analysis. The stability results of oligomer for MVGGVV‐1 suggest that the intermediate state should be trimer (3‐0) and tetramer (2‐2). These methods can be used in stabilization study of other amyloid fibril. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 578–586, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational stability of the full‐atom hexameric model of the ClpB chaperone from Escherichia coli

The Escherichia coli heat shock protein ClpB, a member of the Hsp100 family, plays a crucial role in cellular thermotolerance. In co‐operation with the Hsp70 chaperone system, it is able to solubilize proteins aggregated by heat shock conditions and refold them into the native state in an ATP‐dependent way. It was established that the mechanism of ClpB action depends on the formation of a ring‐shaped hexameric structure and the translocation of a protein substrate through an axial channel. The structural aspects of this process are not fully known. By means of homology modeling and protein–protein docking, we obtained a model of the hexameric arrangement of the full‐length ClpB protein complexed with ATP. A molecular dynamics simulation of this model was performed to assess its flexibility and conformational stability. The high mobility of the “linker” M‐domain, essential for the renaturing activity of ClpB, was demonstrated, and the size and shape of central channel were analyzed. In this model, we propose the coordinates for a loop between b4 and B6 structural elements, not defined in previous structural research, which faces the inside of the channel and may therefore play a role in substrate translocation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 47–60, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural and functional analysis of the Hsp70/Hsp40 chaperone system

As one of the most abundant and highly conserved molecular chaperones, the 70‐kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.     

Development of GMP‐1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aβ) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria‐destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP‐1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP‐1 treatment of SH‐SY5Y cells results in decrease in mitochondria‐associated APP and protects SH‐SY5Y cells from toxic effect of Aβ_1‐42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP‐1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.     

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/α‐synuclein complex by Hip

The ATP‐dependent protein chaperone heat‐shock protein 70 (Hsp70) displays broad anti‐aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α‐synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation‐prone polypeptides. We show a nucleotide‐dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co‐aggregation phenomenon can be prevented in vitro by the co‐chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.     

Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid beta (Abeta) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Abeta aggregation. These observations led to the hypothesis that chaperones might interact with Abeta and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Abeta has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Abeta aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Abeta self-assembly and that these chaperones are effective at substoichiometric concentrations (approximately 1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Abeta is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.     

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.     

Control of cell fate by Hsp70: more than an evanescent meeting

During their lifetime, proteins inevitably expose hydrophobic segments within the polypeptide chains on a molecule's surface, which may be otherwise buried inside the molecules in the proper conformation. This potentially dangerous situation is managed with the aid of the 70-kDa heat shock proteins (Hsp70s) and other molecular chaperones. Although a major function of Hsp70 is assisting in efficient folding of anonymous proteins in unfolded states, recent studies have revealed that Hsp70 plays a variety of specific roles, sometimes deciding the cell fate. These multiple activities are based on the specific binding of Hsp70 to proteins in native states, which regulate cell growth and/or death. It is now well recognized that unfolding of some proteins may cause serious diseases, especially those associated with neurodegeneration, such as Alzheimer's disease. It is suggested that Hsp70 might be a potential drug against these diseases, but caution should be taken because Hsp70 exerts multiple effects by binding to specific proteins.     

Mercury(II) promotes the in vitro aggregation of tau fragment corresponding to the second repeat of microtubule‐binding domain: Coordination and conformational transition

The loss of metal homeostasis and the toxic effect of metal ion are important events in neurodegenerative and age‐related diseases, such as Alzheimer's disease (AD). For the first time, we investigated the impacts of mercury(II) ions on the folding and aggregation of Alzheimer's tau fragment R2 (residues 275‐305: VQIIN KKLDL SNVQS KCGSK DNIKH VPGGGS), corresponding to the second repeat unit of the microtubule‐binding domain, which was believed to be pivotal to the biochemical properties of full tau protein. By ThS fluorescence assay and electron microscopy, we found that mercury(II) dramatically promoted heparin‐induced aggregation of R2 at an optimum molar ratio of 1: 2 (metal: protein), and the resulting R2 filaments became smaller. Isothermal titration calorimetry (ITC) experiment revealed that the strong coordination of mercury(II) with R2 was an enthalpy‐controlled, entropy‐decreased thermodynamic process. The exceptionally large magnitude of heat release (ΔH₁ = ?34.8 Kcal mol^?1) suggested that the most possible coordinating site on the R2 peptide chain was the thiol group of cysteine residue (Cys291), and this was further confirmed by a control experiment using Cys291 mutated R2. Circular dichroism spectrum demonstrated that this peptide underwent a significant conformational change from random coil to β‐turn structure upon its binding to mercury(II) ion. This study was undertaken to better understand the mechanism of tau aggregation, and evaluate the possible role of mercury(II) in the pathogenesis of AD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1100–1107, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Precursors with altered affinity for Hsp70 in their transit peptides are efficiently imported into chloroplasts

Protein import into chloroplasts is postulated to occur with the involvement of molecular chaperones. We have determined that the transit peptide of ferredoxin-NADP(H) reductase precursor binds preferentially to an Hsp70 from chloroplast stroma. To investigate the role of Hsp70 molecular chaperones in chloroplast protein import, we analyzed the import into pea chloroplasts of preproteins with decreased Hsp70 binding affinity in their transit peptides. Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent Km as the wild type precursor and a 2-fold increase in Vmax. Thus, a strong interaction between chloroplast stromal Hsp70 and the transit peptide seems not to be essential for protein import. These results indicate that in chloroplasts the main unfolding force during protein import may be applied by molecular chaperones other than Hsp70s. Although stromal Hsp70s undoubtedly participate in chloroplast biogenesis, the role of these molecular chaperones in chloroplast protein translocation differs from the one proposed in the mechanisms postulated up to date.     

Crystal structure of the nucleotide‐binding domain of mortalin,the mitochondrial Hsp70 chaperone

Mortalin, a member of the Hsp70‐family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe‐S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT‐077. Like other Hsp70‐family members, Mortalin consists of a nucleotide‐binding domain (NBD) and a substrate‐binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide‐binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease‐associated mutation is located on the Mortalin‐NBD surface and may contribute to Mortalin aggregation. We present structure‐based models for how the Mortalin‐NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT‐077. Our structure may contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.     

Multiple Hsp70 Isoforms in the Eukaryotic Cytosol: Mere Redundancy or Functional Specificity?

Hsp70 molecular chaperones play a variety of functions in every organism, cell type and organelle, and their activities have been implicated in a number of human pathologies, ranging from cancer to neurodegenerative diseases. The functions, regulations and structure of Hsp70s were intensively studied for about three decades, yet much still remains to be learned about these essential folding enzymes. Genome sequencing efforts revealed that most genomes contain multiple members of the Hsp70 family, some of which co-exist in the same cellular compartment. For example, the human cytosol and nucleus contain six highly homologous Hsp70 proteins while the yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized Hsp70s. The reasons and significance of the requirement for multiple Hsp70s is still a subject of debate. It has been postulated for a long time that these Hsp70 isoforms are functionally redundant and differ only by their spatio-temporal expression patterns. However, several studies in yeast and higher eukaryotic organisms challenged this widely accepted idea by demonstrating functional specificity among Hsp70 isoforms. Another element of complexity is brought about by specific cofactors, such as Hsp40s or nucleotide exchange factors that modulate the activity of Hsp70s and their binding to client proteins. Hence, a dynamic network of chaperone/co-chaperone interactions has evolved in each organism to efficiently take advantage of the multiple cellular roles Hsp70s can play. We summarize here our current knowledge of the functions and regulations of these molecular chaperones, and shed light on the known functional specificities among isoforms.     

Identifying protein stabilizing ligands using GroEL

Over the past 5 years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (～30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called orphan protein folding diseases. The development of a broad‐based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic, and medical professions. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 237–251, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

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.