Global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.

YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet-undefined function in the oxidative-stress response. YajL protects cells against oxidative-stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. Compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA repair proteins, RNA chaperones, and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative-stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.

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.

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.

DNA replication defects in a mutant deficient in the thioredoxin homolog YbbN.

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, that displays both redox and chaperone properties. Since three out of the six proteins of the YbbN interactome (Butland et al., 2005) are components of DNA polymerase 3 holoenzyme (i.e. the ß-clamp DnaN, the θ subunit HolE and the δ' subunit HolB), we investigated whether the ybbN mutant presents DNA replication defects. We found that this mutant incorporates (3)H-thymidine at higher rates than the parental strain and displays overinitiation, hypermutator and filamentation phenotypes with the occurrence of anucleated cells. Moreover, YbbN functions as a bona fide chaperone in the refolding of the urea-unfolded ß-clamp. These results suggest that the DNA replication and cell division defects of the ybbN mutant might best be explained by chaperone functions of YbbN in the biogenesis of DNA polymerase 3 holoenzyme.

Translational defects in a mutant deficient in YajL, the bacterial homolog of the parkinsonism-associated protein DJ-1.

We report here that YajL is associated with ribosomes and interacts with many ribosomal proteins and that a yajL mutant of Escherichia coli displays decreased translation accuracy, as well as increased dissociation of 70S ribosomes into 50S and 30S subunits after oxidative stress.

Protein aggregation in a mutant deficient in yajL, the bacterial homolog of the Parkinsonism-associated protein DJ-1.

YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase beta subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant.

The thioredoxin homolog YbbN functions as a chaperone rather than as an oxidoreductase.

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxins, and possesses a 20kDa C-terminal part of unknown function. We reported previously that YbbN displays both protein oxido-reductase and chaperone properties in vitro. In this study, we show that an ybbN-deficient strain displays an increased sensitivity to thermal stress but not to oxidative stress, a normal redox state of its cellular proteins but a decreased expression of several cytoplasmic proteins, including EF-Tu, DnaK, GroEL, trigger factor and several Krebs cycle enzymes, suggesting that the chaperone properties of YbbN are more important in vivo than its redox properties. YbbN specifically interacts with DnaK and GroEL, as shown by reverse purification. It increases 4-fold the rate of protein renaturation in vitro by the DnaK chaperone machine, suggesting that it cooperates with DnaK for the optimal expression of several cytoplasmic proteins.

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.