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Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein, Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae*

  • Kondalarao Bankapalli
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India
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  • SreeDivya Saladi
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India
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  • Sahezeel S. Awadia
    Footnotes
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India
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  • Arvind Vittal Goswami
    Footnotes
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India
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  • Madhuja Samaddar
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India
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  • Patrick D'Silva
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Indian Inst. of Science, Biological Sciences Bldg., Bangalore, Karnataka 560012, India. Tel.: 91-080-22932821; Fax: 91-080-23600814.
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India
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  • Author Footnotes
    * This work was supported by a Swarnajayanthi fellowship from the Department of Science and Technology (Grant ID: DST/SJF/LSA-01/2011–2012) and the Department of Biotechnology, India (to P. D.) and by a senior research fellowship from the Council of Scientific and Industrial Research, India (to K. B., A. V. G., and M. S.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Current address: Dept. of Biological Science, University of Toledo, Toledo, OH 43606.
    2 Current address: Dept. of Pathology, Yale School of Medicine, New Haven, CT 06520.
Open AccessPublished:September 14, 2015DOI:https://doi.org/10.1074/jbc.M115.673624
      Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.

      Introduction

      The key metabolic pathways are highly conserved among all eukaryotic organisms that utilize both glycolytic and mitochondrial pathways for normal cellular functions (
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      Cellular metabolic stress: considering how cells respond to nutrient excess.
      ). Although these metabolic pathways are known to be tightly regulated, there is increasing evidence that many metabolites have detrimental effects upon accumulation within the cell (
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      ). Among those, glyoxal and methylglyoxal (MG)
      The abbreviations used are: MG
      methylglyoxal
      ROS
      reactive oxygen species
      PD
      Parkinson disease
      Ni2+-NTA
      nickel-nitrilotriacetic acid
      NAC
      N-acetylcysteine
      MTS
      mitochondrial targeting sequence
      YPD
      yeast extract-peptone-dextrose.
      are potent α-ketoaldehydes that are primarily formed as by-products of glucose metabolism or “glycolysis” in all living organisms (
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      ). The other sources of glyoxal generation are from the enzymatic conversion of intermediates during fatty acid and amino acid metabolism (
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      A systematic approach to evaluate the modification of lens proteins by glycation-induced crosslinking.
      ), collectively referred to as reactive carbonyl species. The reactive carbonyl groups of these glyoxals react indiscriminately with the arginine and lysine residues of proteins, leading to the formation of advanced glycation end-products (
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      ,
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      ). As a result of nucleophilic addition, glyoxals irreversibly modify proteins, including various cellular antioxidant enzymes such as glutathione reductase, glutathione peroxidase, and catalase, leading to significant risks of oxidative stress (
      • Shangari N.
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      The cytotoxic mechanism of glyoxal involves oxidative stress.
      ). The toxicity of these reactive aldehydes is limited not only to the proteome but also extends to lipids and nucleic acids (
      • Thornalley P.J.
      Pharmacology of methylglyoxal: formation, modification of proteins and nucleic acids, and enzymatic detoxification: a role in pathogenesis and antiproliferative chemotherapy.
      ,
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      DNA modifications by the mutagen glyoxal: adduction to G and C, deamination of C and GC, and GA cross-linking.
      ). Organisms possess appropriately evolved detoxifying pathways to neutralize the toxic effects of these harmful metabolites. The detoxification of glyoxals is achieved by two independent mechanisms: first by methylglyoxal reductase enzyme, where MG is converted into d-lactic acid (
      • Murata K.
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      Metabolism of 2-oxoaldehyde in yeasts: purification and characterization of NADPH-dependent methylglyoxal-reducing enzyme from Saccharomyces cerevisiae.
      ); and second, by the glyoxalase systems, comprising two glutathione-dependent enzymes (glyoxalase I and II) that efficiently convert MG into d-lactic acid (
      • Thornalley P.J.
      The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life.
      ).
      The accumulation of advanced glycation end-products and higher levels of glyoxals generates an elevation in the reactive oxygen species (ROS) leading to acute oxidative stress conditions. Although ROS is known to be harmful to cellular constituents, low levels of ROS act as signaling molecules in various pathways, including cell development and division (
      • Fleury C.
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      Mitochondrial reactive oxygen species in cell death signaling.
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      ). Mitochondria are important centers of ROS generation through the complexes of the respiratory electron transport chain (
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      ). In addition, other pro-oxidants such as NADPH oxidase, xanthine oxidase, glucose oxidase, and lipoxygenase are involved in redox reactions contributing to the elevation of ROS in the cell (
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      Bioenergetics and the formation of mitochondrial reactive oxygen species.
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      The genetics and pathology of oxidative phosphorylation.
      ). In a healthy cellular milieu, the ROS levels are stringently regulated by the action of various enzymatic or non-enzymatic antioxidant systems, including catalase, superoxide dismutase, glutathione, and thioredoxins (
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      The changing faces of glutathione, a cellular protagonist.
      ). Imbalance in the ROS homeostasis generates oxidative stress resulting in damage to cellular macromolecules like proteins, lipids, and nucleic acids, which cause aberrations in cellular functions (
      • Fleury C.
      • Mignotte B.
      • Mignotte J.L.
      Mitochondrial reactive oxygen species in cell death signaling.
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      Reactive oxygen species in cell signaling.
      ,
      • Storz G.
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      Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium.
      ). Additionally, acute oxidative stress also causes organellar damage leading to organellar dysfunction (
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      Transient oxidative stress damages mitochondrial machinery inducing persistent beta-cell dysfunction.
      ). Importantly, mitochondrial dysfunction is one of the prominent cellular etiologies associated with premature aging, cardiovascular and retinal disorders, atherosclerosis, and several neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington diseases (
      • Lin M.T.
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      Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.
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      • Dai D.F.
      • Chiao Y.A.
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      Mitochondria in Huntington's disease.
      ,
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      Huntington's disease.
      ).
      Parkinson disease (PD), the second most prevalent age-related neurodegenerative disorder, is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta (
      • Davie C.A.
      A review of Parkinson's disease.
      ). Although PD is predominantly sporadic in nature, mutations in at least five gene loci are implicated in the development of familial PD (
      • Cookson M.R.
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      Parkinson's disease: insights from pathways.
      ,
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      • Hattori N.
      • Murata M.
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      • Toda T.
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      ). These include SNCA (encoding α-synuclein), PARK7 (encoding DJ-1), PARK2 (encoding parkin), PINK1 (encoding Pink1), and LRRK2 (encoding leucine-rich repeat kinase 2) (
      • Davie C.A.
      A review of Parkinson's disease.
      ,
      • Cookson M.R.
      • Bandmann O.
      Parkinson's disease: insights from pathways.
      ,
      • Goswami A.V.
      • Samaddar M.
      • Sinha D.
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      • D'Silva P.
      Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease.
      ,
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      Parkinson's disease: mechanisms and models.
      ,
      • Sulzer D.
      Multiple hit hypotheses for dopamine neuron loss in Parkinson's disease.
      ). Among these genes, DJ-1 belongs to the ThiJ/DJ-1/PfpI superfamily of proteins involved in a plethora of functions such as transcriptional regulation, mitochondrial complex stabilization, and RNA binding (
      • Chen J.
      • Li L.
      • Chin L.S.
      Parkinson disease protein DJ-1 converts from a zymogen to a protease by carboxyl-terminal cleavage.
      ,
      • Shendelman S.
      • Jonason A.
      • Martinat C.
      • Leete T.
      • Abeliovich A.
      DJ-1 is a redox-dependent molecular chaperone that inhibits α-synuclein aggregate formation.
      ,
      • Zhou W.
      • Zhu M.
      • Wilson M.A.
      • Petsko G.A.
      • Fink A.L.
      The oxidation state of DJ-1 regulates its chaperone activity toward α-synuclein.
      ,
      • Xu J.
      • Zhong N.
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      • Kim C.Y.
      • Woldman I.
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      • Gygi S.P.
      • Geula C.
      • Yankner B.A.
      The Parkinson's disease-associated DJ-1 protein is a transcriptional co-activator that protects against neuronal apoptosis.
      ,
      • van der Brug M.P.
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      • Hao L.Y.
      • Lal A.
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      • Cai H.
      • Bonini N.M.
      • Gorospe M.
      • Cookson M.R.
      RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways.
      ). Additionally, DJ-1 has been shown recently to function as an oxidative stress sensor, and its absence leads to increased risk of oxidative stress in dopaminergic neurons of the substantia nigra (
      • Kim R.H.
      • Smith P.D.
      • Aleyasin H.
      • Hayley S.
      • Mount M.P.
      • Pownall S.
      • Wakeham A.
      • You-Ten A.J.
      • Kalia S.K.
      • Horne P.
      • Westaway D.
      • Lozano A.M.
      • Anisman H.
      • Park D.S.
      • Mak T.W.
      Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress.
      ). The homologs of DJ-1 are reported across other species, including Drosophila and Caenorhabditis elegans (
      • Chen P.
      • DeWitt M.R.
      • Bornhorst J.
      • Soares F.A.
      • Mukhopadhyay S.
      • Bowman A.B.
      • Aschner M.
      Age- and manganese-dependent modulation of dopaminergic phenotypes in a C. elegans DJ-1 genetic model of Parkinson's disease.
      ,
      • Menzies F.M.
      • Yenisetti S.C.
      • Min K.T.
      Roles of Drosophila DJ-1 in survival of dopaminergic neurons and oxidative stress.
      ). In contrast to humans, Drosophila has two homologs, namely DJ-1α and DJ-1β (
      • Menzies F.M.
      • Yenisetti S.C.
      • Min K.T.
      Roles of Drosophila DJ-1 in survival of dopaminergic neurons and oxidative stress.
      ). Mutations in both, or in DJ-1β alone, cause a Parkinsonian-like syndrome in Drosophila, characterized by increased sensitivity to oxidative stress and decreased mobility (
      • Meulener M.
      • Whitworth A.J.
      • Armstrong-Gold C.E.
      • Rizzu P.
      • Heutink P.
      • Wes P.D.
      • Pallanck L.J.
      • Bonini N.M.
      Drosophila DJ-1 mutants are selectively sensitive to environmental toxins associated with Parkinson's disease.
      ,
      • Park J.
      • Kim S.Y.
      • Cha G.H.
      • Lee S.B.
      • Kim S.
      • Chung J.
      Drosophila DJ-1 mutants show oxidative stress-sensitive locomotive dysfunction.
      ). The members of ThiJ/DJ-1/PfpI superfamily are also reported in lower eukaryotes, including bacterial species. In Escherichia coli, it is referred to as Hsp31 and is shown to perform multiple functions, including holdase chaperone activity, acid resistance, and aminopeptidase activity of broad specificity (
      • Subedi K.P.
      • Choi D.
      • Kim I.
      • Min B.
      • Park C.
      Hsp31 of Escherichia coli K-12 is glyoxalase III.
      ,
      • Mujacic M.
      • Bader M.W.
      • Baneyx F.
      Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions.
      ,
      • Mujacic M.
      • Baneyx F.
      Chaperone Hsp31 contributes to acid resistance in stationary-phase Escherichia coli.
      ). On the other hand, in yeast, there are four homologs (Hsp31, Hsp32, Hsp33, and Hsp34) belonging to the ThiJ/DJ-1/PfpI family, also classified as the Hsp31 mini-family, for which the functional diversity is poorly understood. Some recent studies indicate that yeast Hsp31 proteins are required against extraneous stress and during the diauxic shift for survival under stationary phase conditions (
      • Miller-Fleming L.
      • Antas P.
      • Pais T.F.
      • Smalley J.L.
      • Giorgini F.
      • Outeiro T.F.
      Yeast DJ-1 superfamily members are required for diauxic-shift reprogramming and cell survival in stationary phase.
      ,
      • Skoneczna A.
      • Miciałkiewicz A.
      • Skoneczny M.
      Saccharomyces cerevisiae Hsp31p, a stress response protein conferring protection against reactive oxygen species.
      ,
      • Wilson M.A.
      Metabolic role for yeast DJ-1 superfamily proteins.
      ).
      Although human DJ-1 is the most extensively studied protein in this superfamily in connection with PD pathology, the functional significance of Hsp31 and its paralogs in yeast is still elusive. In the current study, we have demonstrated the importance of Hsp31 mini-family proteins in protecting cells against oxidative stress induced by MG. Hsp31 exhibits robust GSH-independent glyoxalase activity, and it is critical for glyoxal detoxification as well as suppression of ROS levels. Importantly, human DJ-1 complements the growth of yeast under oxidative and glyoxal stress conditions, signifying its functional conservation across species. Mechanistically, our findings show that Hsp31 maintains GSH and NADPH homeostasis thereby protecting mitochondrial integrity. In summary, our report highlights the specific importance of yeast Hsp31 mini-family proteins in the maintenance of redox homeostasis by modulating cellular antioxidant levels.

      Discussion

      Our present study has uncovered the ROS regulatory role of yeast Hsp31 class of proteins in suppressing the oxidative stress induced by accumulation of toxic metabolic intermediates such as MG. Using yeast genetic and biochemical analyses, we have revealed the vital mechanistic and molecular details of Hsp31 in combating oxidative stress generated by elevation in the levels of glyoxals and reactive oxygen species.

      Robust Methylglyoxalase Activity of Hsp31 Protects against Cellular Damages by Glyoxals and Oxidative Stress in S. cerevisiae

      Although organisms comprise multiple types of glyoxalases for the efficient detoxification of MG accumulated during normal metabolic processes, their functions are largely dependent on the availability of GSH as cofactors. Moreover, the availability of GSH is rate-limiting under oxidative stress conditions, and thus cells demand alternate modes of detoxification of MG. In this report, we have highlighted the significance of the yeast Hsp31 class of proteins, members of the ThiJ/DJ-1/PfpI family possessing robust glyoxalase activity for the suppression of MG-mediated toxicity. Interestingly, the glyoxalase activity of Hsp31-class proteins is GSH-independent, hence providing a unique advantage for cells to combat glyoxal stress (Fig. 9 model).
      Figure thumbnail gr9
      FIGURE 9Model depicting mechanistic details of S. cerevisiae Hsp31 protein in regulating cellular oxidative stress. Step 1, the robust glyoxalase activity of Hsp31 ensures the elimination of toxic methylglyoxal species that arise as a by-product of various metabolic pathways. Step 2, Hsp31 is involved in regulating the GSH/GSSG ratio and NADPH homeostasis, which enables the maintenance of redox homeostasis. Step 3, Hsp31 translocates to the mitochondria and also protects the mitochondrial functions by redistributing GSH levels.
      Previous findings reveal that Hsp31 from E. coli exhibits GSH-independent glyoxalase activity, which efficiently converts MG to lactic acid (
      • Subedi K.P.
      • Choi D.
      • Kim I.
      • Min B.
      • Park C.
      Hsp31 of Escherichia coli K-12 is glyoxalase III.
      ). Contrastingly, however, E. coli Hsp31 plays a minor role in combating oxidative stress but provides acid resistance during starvation conditions in E. coli (
      • Mujacic M.
      • Baneyx F.
      Chaperone Hsp31 contributes to acid resistance in stationary-phase Escherichia coli.
      ). On the other hand, human DJ-1 protein, a member of the ThiJ/DJ-1/PfpI family implicated in familial PD, displays redox-based chaperone activity as well as methylglyoxalase activity (
      • Shendelman S.
      • Jonason A.
      • Martinat C.
      • Leete T.
      • Abeliovich A.
      DJ-1 is a redox-dependent molecular chaperone that inhibits α-synuclein aggregate formation.
      ,
      • Lee J.Y.
      • Song J.
      • Kwon K.
      • Jang S.
      • Kim C.
      • Baek K.
      • Kim J.
      • Park C.
      Human DJ-1 and its homologs are novel glyoxalases.
      ). Because of the conservation of these family proteins across species, we have demonstrated here that YDR533Cp (Hsp31) of S. cerevisiae, a member of the novel ThiJ/DJ-1/PfpI family, possesses robust glyoxalase activity and efficiently suppresses glyoxal toxicity. Besides, the yeast genome encodes for three additional paralogs, namely Hsp32, Hsp33, and Hsp34, which are highly identical in nature but possess a ∼40-fold lower glyoxalase activity as compared with Hsp31 and are hence unable to suppress glyoxal toxicity. Mammalian DJ-1 is known to regulate oxidative stress and reduce ROS levels in dopaminergic neurons. Our results show that deletion of Hsp31 leads to enhancement of the overall cellular ROS and mitochondrial superoxide levels. On the contrary, overexpression of WT Hsp31 restores the ROS to basal levels and suppresses mitochondrial oxidative stress, thus playing an important role in redox homeostasis. Moreover, our results provide the first experimental evidence revealing that human DJ-1 possesses canonical functions and could suppress glyoxal toxicity as well as the oxidative stress phenotype of Hsp31 mutants, thereby signifying the functional conservation of the ThiJ/DJ-1/PfpI family across phylogenetic boundaries. The predicted catalytic triad amino acids, namely Cys-138, His-139, and Glu-170, were found to be very critical for the glyoxalase and ROS regulative functions of Hsp31. On the other hand, the catalytic mutants of human DJ-1, C106A and L166P, which are implicated in familial PD, failed to restore the ROS regulatory effect in Hsp31 deletion cells, suggesting that glyoxalase activity is indispensable for its function across species. Based on our findings, we therefore hypothesized that, unlike E. coli, the ThiJ/DJ-1/PfpI family proteins play a major role in combating oxidative stress and maintaining ROS homeostasis in eukaryotes (Fig. 9 model).

      Hsp31 Maintains Glutathione and NADPH Homeostasis to Combat Oxidative Stress

      The maintenance of steady-state levels of reduced glutathione is critical for cells to survive against oxidative stress-mediated damages. In yeast, the biosynthesis of reduced glutathione is a regulated process requiring two important energy-utilizing enzymes, namely γ-glutamyl-cysteine synthase (Gsh1) and glutathione synthetase (Gsh2) (
      • Penninckx M.J.
      An overview on glutathione in Saccharomyces versus non-conventional yeasts.
      ). At the same time, oxidized glutathione (GSSG) formed during ROS scavenging is recycled to its reduced state by glutathione reductase enzyme (Glr1), which requires NADPH as a cofactor (
      • Grant C.M.
      • Collinson L.P.
      • Roe J.H.
      • Dawes I.W.
      Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation.
      ). Under stress conditions, the activity of key biosynthetic enzymes and the levels of protective antioxidants such as GSH are regulated by several transcription factors, including Nrf2, the activity of which is modulated by ThiJ/DJ-1/PfpI family proteins (
      • Clements C.M.
      • McNally R.S.
      • Conti B.J.
      • Mak T.W.
      • Ting J.P.
      DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2.
      ,
      • Im J.Y.
      • Lee K.W.
      • Woo J.M.
      • Junn E.
      • Mouradian M.M.
      DJ-1 induces thioredoxin 1 expression through the Nrf2 pathway.
      ).
      Our findings provide evidence showing that yeast Hsp31 plays an important role in glutathione homeostasis. The deletion of Hsp31 leads to a decrease in the GSH levels, and overexpression restores intracellular glutathione levels. As a result, the presence of Hsp31 provides cytoprotection to cells by robustly suppressing the cytosolic and mitochondrial ROS levels under both MG and H2O2 stress conditions. Human DJ-1 was shown to up-regulate intracellular glutathione levels by increasing glutamate-cysteine ligase mRNA levels during oxidative stress (
      • Zhou W.
      • Freed C.R.
      DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T α-synuclein toxicity.
      ). Intriguingly, our results show that levels of glutathione biosynthetic enzymes Gsh1 and Gsh2 and recycling enzyme Glr1 are relatively unaffected by Hsp31expression in yeast. On the other hand, during oxidative stress, Glr1-dependent recycling of GSSG to GSH is a key rate-determining event to counter excessive ROS, which is largely dependent on NADPH availability (
      • Grant C.M.
      • Collinson L.P.
      • Roe J.H.
      • Dawes I.W.
      Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation.
      ) (Fig. 9 model). Intriguingly, glyoxals are known to inhibit various NAD(P)H-generating enzymes, leading to NADPH depletion, a major cause of increased oxidative stress upon glyoxal accumulation (
      • Morgan P.E.
      • Sheahan P.J.
      • Davies M.J.
      Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.
      ). In line with this observation, our results suggest that the deletion of Hsp31 led to a significant reduction in total NADPH levels. At the same time, NADPH levels were significantly restored upon Hsp31 overexpression to the WT. On the basis of our findings, it is reasonable to conclude that the robust glyoxalase activity of Hsp31 reduces glyoxal stress, thereby maintaining the intracellular NADPH and glutathione levels required to combat oxidative stress (Fig. 9 model).

      Hsp31 Protects Mitochondrial Integrity by Its Relocalization and Redistribution of GSH Levels under Oxidative Stress Conditions

      Based on our observations, it is evident that Hsp31 plays two significant roles in preserving mitochondrial function by maintaining its integrity. Foremost, the level of Hsp31 is up-regulated upon oxidative stress to suppress mitochondrial superoxide levels. Enhanced mitochondrial oxidative stress results in its fragmentation via alteration in the mitochondrial dynamics (
      • Wu S.
      • Zhou F.
      • Zhang Z.
      • Xing D.
      Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins.
      ). In agreement with this hypothesis, our studies demonstrate that deletion of Hsp31 leads to fragmentation of mitochondria under oxidative stress. Strikingly, the mitochondrial morphology was restored upon overexpression of Hsp31 in the deletion background, thus signifying its critical role in maintaining organelle integrity. Because Hsp31 regulates total cellular GSH availability, it therefore aids in the redistribution into mitochondria to suppress ROS-mediated damages to the organelle. On the other hand, mitochondrial isoform of Glr1 enzyme may play a crucial role in the recycling of oxidized glutathione into its reduced form, thereby maintaining GSH homeostasis (
      • Outten C.E.
      • Culotta V.C.
      Alternative start sites in the Saccharomyces cerevisiae GLR1 gene are responsible for mitochondrial and cytosolic isoforms of glutathione reductase.
      ). Secondly, Hsp31 is predominantly a cytosolic protein; under oxidative stress, it relocalizes into mitochondria to protect its integrity and function. In humans, DJ-1 is known to stabilize complex I as well as interact with mtHsp70; these are the key components regulating mitochondrial oxidative stress (
      • Hayashi T.
      • Ishimori C.
      • Takahashi-Niki K.
      • Taira T.
      • Kim Y.C.
      • Maita H.
      • Maita C.
      • Ariga H.
      • Iguchi-Ariga S.M.
      DJ-1 binds to mitochondrial complex I and maintains its activity.
      ). Importantly, our study demonstrates that the Cys-138 residue is critical for the mitochondrial translocation property. These results are in agreement with reports on mammalian DJ-1, which is known to translocate to mitochondria upon oxidation of cysteine residue (
      • Canet-Avilés R.M.
      • Wilson M.A.
      • Miller D.W.
      • Ahmad R.
      • McLendon C.
      • Bandyopadhyay S.
      • Baptista M.J.
      • Ringe D.
      • Petsko G.A.
      • Cookson M.R.
      The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.
      ). Recent reports show that MG also is involved in the glycation of mitochondrial proteins leading to inhibition of respiration, which is implicated in several pathological conditions in humans (
      • Ray S.
      • Dutta S.
      • Halder J.
      • Ray M.
      Inhibition of electron flow-through complex I of the mitochondrial respiratory chain of Ehrlich ascites carcinoma cells by methylglyoxal.
      ,
      • Biswas S.
      • Ray M.
      • Misra S.
      • Dutta D.P.
      • Ray S.
      Selective inhibition of mitochondrial respiration and glycolysis in human leukaemic leucocytes by methylglyoxal.
      ,
      • Rosca M.G.
      • Monnier V.M.
      • Szweda L.I.
      • Weiss M.F.
      Alterations in renal mitochondrial respiration in response to the reactive oxoaldehyde methylglyoxal.
      ). Intriguingly, DJ-1 family members from E. coli and humans also possess protein deglycase activity, which repairs and reactivates proteins from glycation (
      • Mihoub M.
      • Abdallah J.
      • Gontero B.
      • Dairou J.
      • Richarme G.
      The DJ-1 superfamily member Hsp31 repairs proteins from glycation by methylglyoxal and glyoxal.
      ,
      • Richarme G.
      • Mihoub M.
      • Dairou J.
      • Bui L.C.
      • Leger T.
      • Lamouri A.
      Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.
      ). Although we have not measured the protein deglycase activity of yeast Hsp31, we speculate that the localization of Hsp31 to mitochondria under oxidative stress conditions may contribute to repairing the mitochondrial proteins from glycation, thus protecting organellar integrity. Collectively, our findings underscore the importance of yeast Hsp31 family proteins in the maintenance of the mitochondrial life cycle, which is a key factor in several pathological conditions such as familial PD, where human DJ-1 is known to play a significant role in disease progression.
      In summary, our reports for the first time highlight the significance of ThiJ/DJ-1/PfpI family proteins in protecting the cells against glyoxal stress, which in turn maintains ROS homeostasis in S. cerevisiae by three different mechanisms, as represented in the “model” figure (Fig. 9). First, the robust glyoxalase activity of Hsp31 reduces glyoxal toxicity by converting MG to a less harmful intermediate (lactic acid), thereby alleviating oxidative stress that arises due to the glycation of enzymatic antioxidants. Second, Hsp31 maintains glutathione and NADPH homeostasis, which facilitates the recycling of reduced GSH for the neutralization of excess ROS. Third, enhanced levels of Hsp31 aids in the translocation and redistribution of GSH into mitochondria to suppress elevated levels of ROS and maintain mitochondrial integrity, thereby providing overall cytoprotection.

      Author Contributions

      P. D., K. B., A. V. G., and M. S. designed the study, analyzed the data, and wrote the paper. K. B., S. S., and S. S. A. performed the experiments. K. B. prepared all the figures. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      Yeast Tim44-specific antibody was a kind gift from Prof. Elizabeth A Craig, University of Wisconsin-Madison. We thank Dr. Mark R. Cookson, NIA, National Institutes of Health, for providing the DJ-1 construct. We also thank Dr. H. S. Athreya and Dr. Garima Jaipuria for their help with NMR spectroscopic studies, and we thank the Indian Institute of Science for use of the flow cytometry facility.

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