YajL, prokaryotic homolog of parkinsonism-associated protein DJ-1, functions as a covalent chaperone for thiol proteome

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.     

YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1, protects cells against protein sulfenylation

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently described the oxidative-stress-dependent aggregation of proteins in yajL mutants and the oxidative-stress-dependent formation of mixed disulfides between YajL and members of the thiol proteome. We report here that yajL mutants display increased protein sulfenic acids levels and that formation of mixed disulfides between YajL and its protein substrates in vivo is inhibited by the sulfenic acid reactant dimedone, suggesting that YajL preferentially forms disulfides with sulfenylated proteins. YajL (but not YajL(C106A)) also forms mixed disulfides in vitro with the sulfenylated form of bovine serum albumin. The YajL-serum albumin disulfides can be subsequently reduced by glutathione or dihydrolipoic acid. We also show that DJ-1 can form mixed disulfides with sulfenylated E. coli proteins and with sulfenylated serum albumin. These results suggest that YajL and possibly DJ-1 function as covalent chaperones involved in the detection of sulfenylated proteins by forming mixed disulfides with them and that these disulfides are subsequently reduced by low-molecular-weight thiols.     

Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine,Arginine, and Lysine Residues

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.     

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones trigger factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by overexpression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroEL or SecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that overexpressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that overexpression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK. The relevance for such a convergence of networks of chaperones and proteases acting directly on EF-Tu to modulate the intracellular rate of protein synthesis in response to protein aggregation is discussed.     

YhbO protects cells against multiple stresses

YhbO is a member of the DJ-1/ThiJ/Pfp1 superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1. A yhbO-disrupted mutant of Escherichia coli is highly sensitive to oxidative, thermal, UV, and pH stresses, and the putative nucleophilic cysteine C104 of YhbO is required for stress resistance. These results suggest that YhbO affects a central process in stress management.     

Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson’s disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation.     

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.     

Hfq modulates the sigmaE-mediated envelope stress response and the sigma32-mediated cytoplasmic stress response in Escherichia coli

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the sigma(S)-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the sigma(E)- and sigma(32)-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the sigma(E)-mediated envelope stress response and are defective in sigma(E)-mediated repression of outer membrane proteins. We also show that the sigma(32)-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the "long-term adaptation" of sigma(32) to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli.     

SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O₂⁻) into oxygen and hydrogen peroxide. It is a Cu²⁺/Zn²⁺ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.     

A Homolog of Bacillus subtilis Trigger Factor in Listeria monocytogenes Is Involved in Stress Tolerance and Bacterial Virulence

Molecular chaperones play an essential role in the folding of nascent chain polypeptides, as well as in the refolding and degradation of misfolded or aggregated proteins. They also assist in protein translocation and participate in stress functions. We identified a gene, designated tig, encoding a protein homologous to trigger factor (TF), a cytosolic ribosome-associated chaperone, in the genome of Listeria monocytogenes. We constructed a chromosomal Δtig deletion and evaluated the impact of the mutation on bacterial growth in broth under various stress conditions and on pathogenesis. The Δtig deletion did not affect cell viability but impaired survival in the presence of heat and ethanol stresses. We also identified the ffh gene, encoding a protein homologous to the SRP54 eukaryotic component of the signal recognition particle. However, a Δffh deletion was not tolerated, suggesting that Ffh is essential, as it is in Bacillus subtilis and Escherichia coli. Thus, although dispensable for growth, TF is involved in the stress response of L. monocytogenes. The Δtig mutant showed no or very modest intracellular survival defects in eukaryotic cells. However, in vivo it showed a reduced capacity to persist in the spleens and livers of infected mice, revealing that TF has a role in the pathogenicity of L. monocytogenes.     

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.