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

Abstract

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.

FIGURE 1.

A, increased sensitivity of the ΔyajL mutant to hydrogen peroxide stress. Bacteria (wild-type strain, filled symbols; ΔyajL mutant, open symbols) were incubated for 30 min in the presence of the indicated hydrogen peroxide concentrations, and viable cells were counted. Results are the averages ± S.E. (error bars) of three experiments. B, protein aggregates of the ΔyajL mutant. Insoluble proteins were separated by SDS-PAGE, detected by Coomassie Blue, staining and characterized by N-terminal sequencing. wt, wild-type. C, insoluble proteins were separated by two-dimensional gel electrophoresis as described in Ref. , detected by Coomassie Blue staining, and characterized by mass spectrometry. D, 8,000 × g pellet (lane 1) from the ΔyajL mutant, lysed in the absence of detergent, resuspended in either 50 mm Tris, pH 8, 5 mm MgSO₄, or the same buffer supplemented with 1% Triton X-100 or 5% SDS and incubated for 30 min at 22 °C. The 8,000 × g supernatants of the three samples (buffer alone, lane 2; buffer supplemented with 1% Triton X-100, lane 3; buffer supplemented with 5% SDS, lane 4) were analyzed by SDS-PAGE. ImageQuant software (Molecular Dynamics) was used to quantify proteins.

FIGURE 2.

A, insoluble proteins extracted from bacteria and analyzed by SDS-PAGE: ΔyajL mutant (lane 1); ΔyajL mutant carrying pCA24N-yajL (lane 2), pCA24N-yajLC106A (lane 3), or pCA24N-yajLC106D (lane 4). The amount of protein aggregate in each fraction (quantified using ImageQuant software) is indicated below each lane and is expressed as the percentage of total protein. B, protein aggregation in anaerobic cells. Cultures were grown under anaerobiosis until an A₆₀₀ = 0.5 was reached, and aggregated proteins were analyzed by SDS-PAGE. C, decrease in protein aggregation in the aerobically grown ΔyajL mutant carrying plasmid psodA. Insoluble proteins extracted from the ΔyajL mutant and the ΔyajL mutant carrying plasmid psodA were analyzed by SDS-PAGE. D, exponential phase cells incubated for 20 min in the presence of increasing hydrogen peroxide concentrations ranging from 0 to 40 mm. Protein aggregates were analyzed by SDS-PAGE. The amount of protein aggregates (expressed as a percentage of total protein) is indicated below each lane. E, protein aggregates from the wild-type strain (filled symbols) and ΔyajL mutant (open symbols) (quantified by using ImageQuant software (Molecular Dynamics)) presented as a function of hydrogen peroxide concentration. Results are the averages ± S.E. (error bars) of three experiments.

FIGURE 3.

A, carbonylation levels in protein extracts from the ΔyajL mutant. Wild-type (filled symbols) and yajL-deficient cells (open symbols) were incubated for 20 min in the presence of increasing hydrogen peroxide concentrations. Protein extracts were prepared, spotted onto nitrocellulose membranes, and assayed for protein carbonyls with the OxyBlot kit; carbonyl levels quantified with ImageQuant software are presented as a function of hydrogen peroxide concentration. Results are the averages ± S.E. (error bars) of three experiments. B, protein extracts treated with the OxyBlot kit, separated by SDS-PAGE, transferred to nitrocellulose membranes, and assayed for protein carbonyls.

FIGURE 4.

Bacteria were incubated for 20 min in the absence or presence of 2 mm hydrogen peroxide, and insoluble proteins were extracted from the parental strains BW25113 (wt₁, parental strain of the ΔyajL mutant) and MG1655 (wt₂, parental strain of the skx and hpx mutants), as well as from the yajL, skx, and hpx mutants, separated by SDS-PAGE and detected by Coomassie Blue staining.

FIGURE 5.

Chaperone properties of YajL. A, citrate synthase renaturation. Citrate synthase was denatured in urea and then renatured for 20 min by dilution as described under “Experimental Procedures,” at a concentration of 0.1 μm, either in the absence of additional protein (control) or in the presence of 2 μm reduced YajL, 2 μm DnaK, 1 μm DnaK/0.4 μm DnaJ/0.4 μm GrpE, 0.2 mm ATP, and 2 mm MgCl₂, or 2 μm YhbO. B, dependence of citrate synthase renaturation on the concentrations of reduced or oxidized YajL. Citrate synthase (0.1 μm) was renatured in the presence of reduced (filled circles) or oxidized (open circles) YajL at the indicated concentrations. C, dependence of ribosomal proteins S1 and L3 solubilization on reduced or oxidized YajL. S1 (circles) and L3 (squares) were denatured in urea and subsequently diluted for 20 min in buffer containing reduced (filled symbols) or oxidized (open symbols) YajL at the indicated concentrations. Samples were centrifuged for 10 min at 15,000 × g, and supernatants and pellets were analyzed by SDS-PAGE. The amounts of S1 and L3 in the supernatant fractions are shown. Ribosomal protein L3 in supernatants and pellets (silver-stained) from the solubilization experiment performed with oxidized YajL (open squares) is shown above the figure.

FIGURE 6.

mRNA levels in the ΔyajL mutant. Northern blot analysis of OmpA (A), FliC (B), L1 (C), and S1 (D) mRNAs prepared from the wild-type strain (filled symbols) and the ΔyajL mutant (open symbols), grown in LB medium at 37 °C to midexponential phase. Hybridization detected the 1041-nucleotide ompA mRNA, the 1497-nucleotide fliC mRNA, the 2352-nucleotide bicistronic cmk rpsA mRNA, and the 1128-nucleotide bicistronic rplK rplA mRNA (supplemental Fig. S7). We quantified the mRNA bands on Northern blots using National Institutes of Health 1.62 software and plotted the mRNA levels as a function of the time elapsed after exposure of bacteria to 0.3 mm hydrogen peroxide. A ssrA-specific probe (tmRNA) was used as a control for the normalization of each sample. The results are normalized to the amounts (before oxidative stress) of the ompA, rplK rplA (L1), cmk rpsA (S1) mRNAs in the wild-type strain and fliC mRNA in the mutant strain. The results are the mean values of three independent experiments. Results are the averages ± S.E. (error bars) of three experiments.