L166P mutant DJ-1 promotes cell death by dissociating Bax from mitochondrial Bcl-X_L

Considering that DJ-1 and its pathogenic mutant DJ-1(L166P) have potential functions in mitochondria, we first examined the subcellular localization of DJ-1 and DJ-1(L166P) in HEK293 cells. DJ-1-Myc was distributed diffusely in both the cytoplasm and nucleus, with a small portion co-localized with MitoTracker (green) (Figure 1A and 1B). However, DJ-1(L166P)-Myc was dominantly presented in the mitochondria with much less nuclear and cytosolic distribution (Figure 1A). Quantitative analysis showed that approximately 81.3% of cells transfected with DJ-1(L166P) displayed a mitochondrial localization, and approximately 18.7% of them displayed a cytosolic localization (Figure 1B). Consistent with the immunocytochemical results, subcellular fractionation assays also showed that both of distribution ratio and protein level of DJ-1(L166P) in the mitochondrial fraction were much higher than those of wild-type DJ-1, although the total protein level of DJ-1(L166P) was much less than that of wild-type DJ-1 (Figure 1C and 1D). The lower level of Flag-DJ-1(L166P) protein compared to Flag-DJ-1 should be caused by that L166P mutant is unstable and degraded rapidly through the ubiquitin proteasome system (UPS), when equal amounts of plasmids are used for transfection [6, 7, 38]. The mitochondrial localization of wild-type DJ-1 and its mutants increases under oxidative stresses such as paraquat treatment,H₂O₂ and UV irradiation [34, 36]. Consistent with those findings, we observed that UVB irradiation increased the mitochondrial localization of both endogenous DJ-1 and Flag-DJ-1(L166P), but did not change total protein levels of them (Figure 1E and 1F). These results indicated that DJ-1(L166P) is prone to mitochondrial localization and the mitochondrial distribution of wild-type DJ-1 and DJ-1(L166P) are increased in response to UVB irradiation.

In our previous study, we showed that wild-type DJ-1 translocates to mitochondria to bind to Bcl-X_L in response to UVB irradiation [37]. Considering that DJ-1(L166P) is mainly distributed in mitochondria, and translocates more to mitochondria under oxidative stress, we wonder whether DJ-1(L166P) binds to Bcl-X_L. Although the interactions of wild-type DJ-1 and DJ-1(L166P) with Bcl-X_L were not significant different in GST pulldown assays in vitro (Figure 2A), more DJ-1(L166P) than wild-type DJ-1 bound to Bcl-X_L in cells (Figure 2B). Neither wild-type DJ-1 nor DJ-1(L166P) bound to Bcl₂ and Bax, another two typical Bcl-2 family proteins (data not shown). These data suggested that wild-type DJ-1 and DJ-1(L166P) specifically bind to Bcl-X_L. The monoclonal anti-Bcl-X_L antibody used in Figure 2B is suitable for immunoprecipitation assays as Flag-Bcl-X_L could be immunoprecipitated by anti-Bcl-X_L antibody but not by control mouse serum IgG ( Additional file 1: Figure S1). Consistent with data from immunoprecipitation analyses, immunocytochemical studies showed that DJ-1(L166P)-Myc, but not DJ-1-Myc, was well co-localized with EGFP-Bcl-X_L in HEK293 cells (Figure 2C). We also examined the interactions between Bcl-X_L and another pathogenic DJ-1 mutant, DJ-1(M26I). Similar to DJ-1(L166P), DJ-1(M26I) interacted with Bcl-X_L and co-localized with Bcl-X_L ( Additional file 1: Figure: S2). As DJ-1(L166P) increased in mitochondria under UVB irradiation (Figure 1E and 1F), we next performed immunoprecipitation assays to test if the interaction of Bcl-X_L with DJ-1(L166P) is affected by UVB irradiation. Interestingly, the binding affinity of Flag-DJ-1(L166P) for EGFP-Bcl-X_L significantly increased after UVB irradiation (Figure 2D). In addition, UVB irradiation led to larger punctate DJ-1(L166P)-RFP spots co-localizing with EGFP- Bcl-X_L (Figure 2E). Moreover, the mitochondria exhibited more severe abnormalities in cells harboring DJ-1(L166P) under UVB irradiation (Figure 2E).

We previously found that wild-type DJ-1 mainly binds to amino acids 86–195 of Bcl-X_L which contain BH1, BH2 and BH3 domains [37]. We wonder whether DJ-1(L166P) binds to the same amino acids of Bcl-X_L. Surprisingly, DJ-1(L166P) bound to the C-terminal fragment of Bcl-X_L at amino acids 196–233 (Figure 3A). We further examined the regions of Bcl-X_L binding to DJ-1(L166P) in H1299 cells. Similar to the results from GST pulldown assays, EGFP-Bcl-X_L^196-233 but not EGFP-Bcl-X_L^1-195 was found to interact with Flag-DJ-1(L166P) (Figure 3B). These results suggest that DJ-1(L166P) and wild-type DJ-1 bind to different domains of Bcl-X_L, indicating that they may regulate Bcl-X_L functions differently. Under UVB irradiation, Bcl-X_L is degraded via the UPS [39, 40]. In our previous study, we showed that wild-type DJ-1 stabilizes Bcl-X_L by its inhibiting Bcl-X_L under UVB irradiation. We therefore examined if DJ-1(L166P) also stabilize Bcl-X_L. Under UVB irradiation, knockdown of DJ-1 decreased Bcl-X_L protein levels and re-overexpression of Flag-DJ-1(s), a synonymous mutant that is resistant to si-DJ-1, restored Bcl-X_L protein levels, however, Flag-DJ-1(L166P)(s) did not (Figure 3C and 3D). Meanwhile, the ubiquitination of Bcl-X_L was inhibited by DJ-1 but not DJ-1(L166P) (Figure 3E).

Bcl-2 family proteins mediate apoptosis in a manner dependent on their homo- or hetero-dimerization [41]. Bcl-X_L interacts with Bax to block its oligomerization in the mitochondrial membrane, thereby protecting cells from Bax-induced mitochondrial membrane permeabilization [41‐43]. It has been reported that the BH1-2 domains and the C-terminus of Bcl-X_L are required for Bcl-X_L/Bax heterodimer formation [43, 44]. To investigate if DJ-1(L166P) affects the interactions between Bcl-X_L and Bax or Bcl-2, we performed competitive binding assays. With less amount of His-DJ-1(L166P), more Bax bound to Bcl-X_L (Figure 4A). However, the binding ability of Bcl-2 to Bcl-X_L was not affected by His-DJ-1(L166P) (Figure 4B). To further identify if DJ-1(L166P) influences the interactions between Bcl-X_L and Bax in mammalian cells, we transfected various amounts of Flag-DJ-1(L166P) into H1299 cells stably expressing EGFP-Bcl-X_L or EGFP-Bcl-X_L^1-195 and performed immunoprecipitation assays. Under UVB irradiation, the amount of endogenous Bax that interacted with EGFP-Bcl-X_L was decreased when more Flag-DJ-1(L166P) was inputted (Figure 4C and 4D). However, EGFP-Bcl-X_L^1-195, which does not interact with Bax [44], was unable to interact with DJ-1(L166P) (Figure 4C).

The mitochondrial localization of Bax is important for its ability to induce cell death [41]. Because DJ-1 and DJ-1(L166P) re-distribute to mitochondria upon UVB irradiation but differentially influence Bcl-X_L, we performed cytosolic and mitochondrial fractionation assays and MTT assays to examine the effects of DJ-1 and DJ-1(L166P) on mitochondrial Bax translocation and cell viability. We performed experiments in H1299 cells, a p53 null cell line to exclude the possibility that DJ-1 inhibits Bax transcription by binding to p53 [45‐47]. Because endogenous DJ-1 expression is abundant [48], we constructed a H1299 cell line stably transfected with sh-DJ-1 to silence endogenous DJ-1 to examine the effects of exogenous wild-type DJ-1 and DJ-1(L166P).

The knockdown efficiency of sh-DJ-1 is shown in Figure 5A and 5B. In the absence of UVB irradiation, neither Flag-DJ-1(s) nor Flag-DJ-1(L166P)(s) had significant effects on the translocation of Bax from the cytosol to the mitochondria in sh-DJ-1 cells (Figure 5C and 5D). However, under UVB irradiation, less Bax was presented in the mitochondrial fraction in cells transfected with Flag-DJ-1(s), but more Bax was present in the mitochondrial fraction in cells transfected with Flag-DJ-1(L166P)(s) (Figure 5C and 5D). Overexpression of Flag-DJ-1(s) but not Flag-DJ-1(L166P)(s) significantly increased mitochondrial Bcl-X_L in response to UVB irradiation (Figure 5C and 5E). In addition, in the absence of death stimulus, overexpression of Flag-DJ-1(s) or Flag-DJ-1(L166P)(s) had no significant effects on Bcl-X_L levels, caspase-3 and PARP cleavage (Figure 5F) or cell viability (Figure 5G). However, with UVB irradiation, Flag-DJ-1(s) partially restored Bcl-X_L levels and accordingly inhibited the cleavage of caspase-3 and PARP (Figure 5F) and increased cell viability (Figure 5G). In contrast to Flag-DJ-1(s), Flag-DJ-1(L166P)(s) greatly increased the cleavage of both caspase-3 and PARP (Figure 5F) and decreased cell viability (Figure 5G). Moreover, the effects of DJ-1 and DJ-1(L166P) on cell death under UVB irradiation were abrogated by Bcl-X_L knockdown (Figure 5F and 5G). These results suggest that wild-type DJ-1 protects cells against UVB irradiation by inhibiting Bcl-X_L degradation, but DJ-1(L166P) promotes cell death by dissociating Bcl-X_L/Bax heterodimerization.

Human HEK293 cells, a human kidney cell line, and H1299 cells, a human lung cancer cell line, were maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (Hyclone, USA). Plasmid transfections were performed using Lipofectamine2000 reagent (Invitrogen, USA).

HEK293 or H1299 cells were irradiated with UVB using a UV crosslinker. Briefly, the cultured cells covered with a thin layer of phosphate buffer solution (PBS, pH 7.4), were exposed to UVB irradiation (312 nm) with 80 mJ/cm2 with a UV crosslinker (SCIENTZ03-II, Ningbo, China). After UVB irradiation, the cells were cultured for 16 hours and subsequently subjected to additional experiments.

si-DJ-1³²⁸ was described previously [47]. siRNA against human Bcl-X_L mRNA was purchased from GenePharma (GenePharma, Shanghai, China) with the following sequences: sense: 5^′-GAGAUGCAGGUAUUGGUGATT-3^′, anti-sense: 5^′-UCACCAAUACCUGCAUCUCTT-3^′. The oligonucleotides were transfected with Oligofectamine reagent (Invitrogen, USA). Briefly, cultured cells were washed with Opti-MEM medium (Invitrogen) and then transfected with siRNA using Oligofectamine reagent in Opti-MEM medium without serum. Six hours after transfection, the culture medium was replaced with fresh complete medium. The cells were subjected to further experiments 72 hours after transfection. pGPU6/GFP/Neo-sh-DJ-1 encoding a short hairpin RNA (shRNA) against nucleotide 328 to 346 of human DJ-1 mRNA (sh-DJ-1) or a negative control short hairpin (sh-NC) was constructed by GenePharma (GenePharma, Shanghai, China). H1299 cells stably expressing sh-NC or sh-DJ-1 were obtained by selection with 200 μg/ml Geneticin (Invitrogen, USA) after transfection.

Full-length DJ-1 in p3 × Flag-myc-cmv-24, pET-15b, pDsRed-N1, pmyc-cmv-24 and pGEX-5x-1, and pET-21a-Bcl₂, pET-21a-Bax, pET-21a-Bcl-X_L, pEGFP-C2-Bcl-X_L, p3 × Flag-myc-cmv-24-Bcl-X_L, pGEX-5x-1-Bcl-X_L(1–85), pGEX-5x-1-Bcl-X_L(86–195) and pGEX-5x-1-Bcl-X_L(196–233) were described previously [37]. pEGFP-C2-Bcl-X_L(1–195) and pEGFP-C2-Bcl-X_L(196–233) were created by subcloning PCR products into pEGFP-C2 at its EcoRI/SalI sites. The PCR products were amplified with the following primers: 5^′-CGGAATTCATGTCTCAGAGCAAC-3^′ and 5^′-GCGTCGACTCAATAGAGTTCCACAAA-3^′ for 1-195aa; 5^′-CGGAATTCGGGAACAATGCAGCA-3^′ and 5^′-GCGTCGACTCATTTCCGACTGAAG-3^′ for 196-233aa. DJ-1(L166P) and DJ-1(M26I) mutants were obtained by site-directed mutagenesis using wild-type DJ-1 plasmids as template with the following primers: 5^′-CCTGCAATTGTTGAAGCCCTGGAATG-3^′ and 5^′-CGCAAACTCGAAGCTGGTCCCAG-3^′, for DJ-1(L166P); 5^′-GTAGATGTCATTAGGCGA-3^′ and 5^′-GGTGACCTTAATCCCAGC-3^′, for DJ-1(M26I); respectively. Two synonymous mutants, p3 × Flag-myc-cmv-24-DJ-1(s) and p3 × Flag-myc-cmv-24-DJ-1(L166P)(s), that are resistant to si-DJ-1³²⁸ and sh-DJ-1 were described previously [54].

HEK293 cells were washed with PBS and fixed with 4% paraformaldehyde. After being blocked with 4% fetal bovine serum containing 0.25% Triton X-100 in PBS, the cells incubated with rabbit anti-myc polyclonal antibodies (Santa Cruz Biotechnology, USA) followed by an incubation with rhodamine-conjugated donkey anti-rabbit IgG (Santa Cruz biotech, Inc). After staining with DAPI (4^′,6-diamidino-2-phenylindole), the labeled cells were observed using an inverted fluorescent microscope (Olympus, IX71).

Equal amounts of GST or GST-fused proteins (20 μg) expressed by Escherichia coli strain JM109 were incubated with 20 μl of glutathione agarose beads (Pharmacia, USA) for 30 min at 4°C. After washing three times with ice-cold PBS, the beads were incubated with 50 μg of His-fused protein expressed by Escherichia coli strain BL21 for 2 hours at 4°C. After incubation, the beads were washed five times with ice-cold HNTG buffer (20 mM Hepes-KOH, pH 7.5, 100 mM NaCl, 0.1% Triton X-100 and 10% glycerol). Bound proteins were eluted from the beads and subjected to immunoblot analysis with specific antibodies. The input represents 10% of the protein that was incubated with GST or a GST-fused protein. The inputs of purified GST and GST fusion proteins are stained with Coomassie Brilliant Blue (CBB) or anti-GST antibody.

The cells were lysed in 1 ml cell lysis buffer (50 mM Tris–HCl pH 7.5 buffer containing 150 mM NaCl, 1% NP40 and 0.5% deoxycholate) supplemented with the protease inhibitor cocktail (Roche, USA) for 30 min at 4°C. After centrifugation at 12,000 g for 15 min at 4°C, the supernatants were incubated with appropriate antibodies coupled to protein G Sepharose (Roche, USA). The immunoprecipitants were then washed five times with cell lysis buffer. Bound proteins and cell lysates were subjected to immunoblot analysis. The input represents 10% of the supernatant used in the co-immunoprecipitation experiment.

Proteins were separated by 12% or 15% SDS-PAGE and subjected to immunoblot analysis with specific antibodies. The following primary antibodies were used: Monoclonal anti-Bcl2, anti-Bcl-XL, anti-GFP, anti-GST, anti-Tom20, anti-Ub and polyclonal anti-Myc, anti-Bax, anti-Max antibodies were purchased from Santa Cruz Biotechnology. Polyclonal anti-Bcl-XL, anti-cleaved caspase-3 and anti-PARP antibodies were from Cell Signaling. Polyclonal anti-DJ-1 antibodies were purchased from Chemicon. Monoclonal anti-Flag-HRP and anti-α-Tubulin antibodies were purchased from Sigma. Monoclonal anti-GAPDH antibody was from Millipore. The secondary antibodies, sheep anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Amersham Pharmacia Biotech. The proteins were visualized using an ECL detection kit (Amersham Pharmacia Biotech). Immunoblot densitometric analysis of data from three independent experiments was performed using Photoshop 7.0 (Adobe, USA).

The cytosolic and mitochondrial fractions were isolated using Mitochondria Isolation Kit for Cultured Cells (Beyotime, China). The total cell lysates and isolated fractions were subjected to immunoblot analysis with specific antibodies. Tom20, α-Tubulin and Max served as the mitochondrial, cytosolic and nuclear maker, respectively.

The cell viability was measured by MTT (methylthiazoletetrazolium) assay. Briefly, the cells were washed with DMEM without phenol red (Gibco, USA) and incubated with 0.5 mg/ml MTT (Sigma) for three hours. The medium was removed and the formazan crystals were dissolved in DMSO (dimethyl sulfoxide). Cell viability was measured by spectrometry at OD570. The data were normalized to a control and the ratios are presented as means ± S.E.M from three independent experiments.

The data were analyzed by one-way analysis of variance (ANOVA) using origin 6.0 software (Originlab, USA). Values are shown as mean ± S.E.M.