PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

Abstract

Macroautophagy is induced under various stresses to remove cytotoxic materials, including misfolded proteins and their aggregates. These protein cargoes are collected by specific autophagic receptors such as SQSTM1/p62 (sequestosome 1) and delivered to phagophores for lysosomal degradation. To date, little is known about how cells sense and react to diverse stresses by inducing the activity of SQSTM1. Here, we show that the peroxiredoxin-like redox sensor PARK7/DJ-1 modulates the activity of SQSTM1 and the targeting of ubiquitin (Ub)-conjugated proteins to macroautophagy under oxidative stress caused by TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10). In this mechanism, TNFSF10 induces the N-terminal arginylation (Nt-arginylation) of the endoplasmic reticulum (ER)-residing molecular chaperone HSPA5/BiP/GRP78, leading to cytosolic accumulation of Nt-arginylated HSPA5 (R-HSPA5). In parallel, TNFSF10 induces the oxidation of PARK7. Oxidized PARK7 acts as a co-chaperone-like protein that binds the ER-derived chaperone R-HSPA5, a member of the HSPA/HSP70 family. By forming a complex with PARK7 (and possibly misfolded protein cargoes), R-HSPA5 binds SQSTM1 through its Nt-Arg, facilitating self-polymerization of SQSTM1 and the targeting of SQSTM1-cargo complexes to phagophores. The 3-way interaction among PARK7, R-HSPA5, and SQSTM1 is stabilized by the Nt-Arg residue of R-HSPA5. PARK7-deficient cells are impaired in the targeting of R-HSPA5 and SQSTM1 to phagophores and the removal of Ub-conjugated cargoes. Our results suggest that PARK7 functions as a co-chaperone for R-HSPA5 to modulate autophagic removal of misfolded protein cargoes generated by oxidative stress.

Keywords: Macroautophagy; N-end rule pathway; N-terminal arginylation; SQSTM1; protein quality control; proteolysis.

Figure 1.

Protective role of PARK7 in TNFSF10-induced oxidative stress. (a) Cells were treated with various concentrations (1–10 ng/ml) of TNFSF10 for 4 h and labeled with MitoSOX Red. The total fluorescence intensity of oxidized MitoSOX Red was measured using flow cytometry. (b) Quantification of A. Error bars represent the mean ± SEM from 3 separate experiments (*p < 0.05). (c) Cells were stably transfected with shScramble or shPARK7 and treated with 10 ng/ml TNFSF10 for 4 h. Mitochondria and cells were stained with MitoTracker Green and MitoSOX Red, respectively. Mitochondria (green) and mitochondrial superoxide anion (red) were detected using confocal microscopy. Scale bar: 10 μm. (d) Cells were stably transfected with shScramble or shPARK7, followed by treatment for 4 h with 2.5 mM NAC, 10 ng/ml TNFSF10 or in combination. Cells were subsequently stained with CM-H₂-DCFDA for 30 min. Green signals indicate ROS in the cytosol. Scale bar: 200 μm. (e) Cells were stably transfected with control shRNA (shScram.) or PARK7 shRNA (shPARK7), followed by the treatment with 10 ng/ml TNFSF10 for 4 h. Cells were subsequently labeled with MitoSOX Red, and MitoSOX Red fluorescence was analyzed using flow cytometry. a.u., arbitrary units. (f) Quantification of E. Error bars represent the mean ± SEM from 3 separate experiments (**p < 0.01).

Figure 2.

PARK7 interacts with R-HSPA5, the Nt-arginylated form of HSPA5. (a) Cells were engineered to stably express either p3x-FLAG or FLAG-tagged PARK7 (FLAG-PARK7) and treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated using anti-FLAG antibody, and the precipitated proteins were visualized using silver staining. (b) Cells were transiently transfected with a plasmid expressing either FLAG-PARK7 or HA-tagged HSPA5. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody (top). The presence of FLAG-PARK7 and HA-HSPA5 in the lysates was verified by immunoblotting (bottom). (c) Cells were treated with 5 ng/ml TNFSF10 for 3 h, and cell lysates were immunoprecipitated with anti-PARK7 antibody or mock antibody (rabbit IgG) followed by immunoblotting with anti-HSPA5 or anti-PARK7 antibody (top). The presence of HSPA5 and PARK7 in the lysates was verified using immunoblotting (bottom). (d) A schematic diagram in which TNFSF10 induces the Nt-arginylation of HSPA5. In this mechanism, newly synthesized HSPA5 translocates into the ER lumen, during which its signal peptide is cleaved off by the signal peptide peptidase, resulting in mature HSPA5. Our results suggest that TNFSF10 induces the cytosolic retrotranslocation and Nt-arginylation of lumenal HSPA5, resulting in cytosolic accumulation of R-HSPA5. (e) HCT116 cells were treated with 10 ng/ml TNFSF10, followed by immunoblotting of R-HSPA5, HSPA5, and ATE1. (f) HCT116 cells were treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were fractionated to enrich the cytosol, mitochondria, and ER. Fractionated proteins were immunoblotted for R-HSPA5, PARK7, HSPA5, the mitochondrial channel VDAC (voltage dependent anion channel), the ER chaperone CANX (calnexin).

Figure 3.

TNFSF10 induces the interaction of oxidized PARK7 with R-HSPA5. (a) HCT116 cells were transfected with a plasmid encoding FLAG or FLAG-PARK7. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. (b) HCT116 cells stably expressing FLAG or FLAG-PARK7 were treated with 5 ng/ml TNFSF10 for 4 h. Cell lysates were incubated with GST-PARK7 proteins for 2 h then immunoprecipitated with glutathione bead, followed by immunoblotting with the indicated antibodies. (c) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-SQSTM1 antibody, followed by immunoblotting analysis. (d) HCT116 cells were treated with 200 nM bafilomycin A₁ (Baf) for 6 h or cultured in the presence of 200 nM bafilomycin A₁ for 2 h then additionally treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-SQSTM1 antibody, followed by immunoblotting analysis. (e) A schematic diagram indicating the interaction between arginylated HSPA5, oxidized PARK7 (oxPARK7), and activated SQSTM1. In this mechanism, HSPA5, PARK7 and SQSTM1 are each modified, and the modification of these 3 components constitutes the complex of 3-way interaction. (f) A schematic diagram in which recombinant Ub-R/V-HSPA5-GFP proteins are processed by a deubiquitination enzyme (DUB). In this mechanism, Ub-R/V-HSPA5-GFP is expressed in HCT116 cells and its ubiquitin is cleaved by DUB and arginine or valine is exposed as a result of cleavage as shown. (g) HCT116 cells were co-transfected with plasmids encoding FLAG-PARK7 and Ub-R-HSPA5-GFP or Ub-V- HSPA5-GFP. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody.

Figure 4.

PARK7 is oxidized by TNFSF10 and the oxidation of PARK7 is required for the interaction of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h, followed by immunoblotting with specific antibodies for oxidized PARK7 and PARK7. (b) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h or co-treated with 10 ng/ml TNFSF10 and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (c) HCT116 cells were treated with 250 μM tert-butyl hydroperoxide (tBHP) for 3 h or co-treated with 250 μM tBHP and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (d) HCT116 cells were co-transfected with plasmids encoding Ub-R-HSPA5-GFP and one of the following: FLAG-tagged wild-type PARK7 or its C46A, C53A, and C106A mutants. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody. (e) HCT116 cells were transfected with a plasmid encoding FLAG, FLAG-PARK7, or FLAG-PARK7^C106A. After 48 h, cells were treated with 20 ng/ml TNFSF10 for 2 h. Cell lysates were immunoprecipitated with the anti-FLAG antibody and then immunoblotted with the indicated antibodies.

Figure 5.

PARK7 is required for the autophagic targeting of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) Cells were treated with 200 nM bafilomycin A₁ for 6 h or 10 ng/ml TNFSF10 for 4 h. Alternatively, the cells cultured in the presence of 200 nM bafilomycin A₁ for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. Immunostaining analysis was performed using antibodies to R-HSPA5 (red) and SQSTM1 (green), followed by confocal microscopy. Scale bar: 10 μm. (b) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 10 ng/ml TNFSF10 for 4 h or 200 nM bafilomycin A₁ (Baf) for 2 h and additionally treated with 10 ng/ml TNFSF10 for 4 h as described in A. (c) Quantification of R-HSPA5 cytosolic puncta in B. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (d) Quantification of SQSTM1 cytosolic puncta in B. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (e) Quantification of the colocalization of SQSTM1 cytosolic puncta in B with R-HSPA5 puncta. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (f) Cells were treated with 10 ng/ml TNFSF10 for 4 h or 200 nM bafilomycin A₁ for 6 h. Alternatively, the cells cultured in the presence of 200 nM bafilomycin A₁ for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. The SQSTM1 and PARK7 oligomerization assay was followed by nonreducing SDS-PAGE and immunoblotting using antibodies to SQSTM1 and PARK7. (g) Cells were treated with tBHP at the indicated concentration. PARK7 oligomerization assay was followed by nonreducing SDS-PAGE and immunoblotting using an antibody to PARK7.

Figure 6.

The autophagic targeting of R-HSPA5 and SQSTM1 is impaired in PARK7-deficient cells. (a) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ (Baf) for 6 h and immunostained for LC3-II (red) or PARK7 (green). LC3-II-positive autophagic vacuoles were examined using confocal microscopy. Scale bar: 10 μm. (b) Quantification of the number of LC3-II puncta in A. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (c) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 2 h, followed by the treatment with 10 ng/ml TNFSF10 for 4 h. The cells were immunostained for LC3-II (red) or SQSTM1 (green). Puncta formation and colocalization of LC3-II and SQSTM1 were examined using confocal microscopy. Scale bar: 10 μm. (d) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 2 h, followed by treatment with 10 ng/ml TNFSF10 for 4 h. The cells were immunostained for PLEKHM1 (green). Puncta formation of PLEKHM1 was examined using confocal microscopy. Scale bar: 10 μm. (e) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ (Baf) for 6 h and immunoblotted with the indicated antibodies. (f) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 6 h and immunostained for WIPI2 (red). WIPI2-positive autophagic vacuoles were examined using confocal microscopy. Scale bar: 10 μm.

Figure 7.

PARK7 is required for the removal of Ub conjugates in HCT116 cells treated with TNFSF10. (a) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with the presence of 200 nM bafilomycin A₁ (Baf) for 2 h and were additionally treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were subjected to immunoblotting analysis using FK2 antibody specific to Ub-conjugated proteins. (b) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 10 ng/ml TNFSF10 for 4 h alone or were pretreated with bafilomycin A₁ (Baf) for 2 h, followed by the treatment with 10 ng/ml TNFSF10 for 4 h, followed by immunostaining analysis using FK2 antibody (red). Scale bar: 10 μm (c) Quantification of the intensity of Ub-positive puncta as visualized using FK2 antibody. Error bars represent the mean ± SEM from each cells (***p < 0.001, n = 50). (d) Quantification of the number of Ub-positive puncta as visualized using FK2 antibody. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (e) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 2 h, followed by treatment with 10 ng/ml TNFSF10 for 4 h. The cells were immunostained using FK2 (red) or SQSTM1 (green) antibodies. Puncta formation and colocalization of FK2 and SQSTM1 signals were examined using confocal microscopy. Scale bar: 10 μm. (f) Same as E except that FK2 (red) or LC3 (green) antibodies were used.

Figure 8.

PARK7 is required for the targeting of KEAP1 to autophagy in HCT116 cells treated with oxidative stressors. (a) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 2 h, followed by treatment with 10 ng/ml TNFSF10 for 4 h. The cells were immunostained using KEAP1 (red) or SQSTM1 (green) antibodies. Puncta formation and colocalization of KEAP1 and SQSTM1 signals were examined using confocal microscopy. Scale bar: 10 μm. (b) HCT116 cells cultured in the presence of 200 nM bafilomycin A₁ (Baf) for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. The cells were immunoblotted for anti-KEAP1 antibody. (c) Cells cultured in the presence of 200 nM bafilomycin A₁ for 3 h were additionally treated with 250 μM tBHP for 3 h. The cells were immunostained for KEAP1 (red) and SQSTM1 (green), and examined using confocal microscopy. Scale bar: 10 μm.

Figure 9.

Hypothetical model for the role of PARK7 in autophagic protein quality control. In this model, TNFSF10 causes mitochondrial misregulation and oxidative stress associated with the excessive generation of ROS. This causes the formation of cytosolic misfolded proteins that are tagged with Ub but cannot be degraded by the UPS (step 1). In response to the proteotoxicity, cells induce autophagic protein quality control, which involves the Nt-arginylation of the ER-resident HSPA5 (step 2). In parallel, PARK7 is oxidized (step 3). The resulting PARK7 binds R-HSPA5 as its cofactor/co-chaperone that facilitates the ability of association with binding Ub-tagged misfolded protein clients (step 4) and enhances the ability of R-HSPA5 to activate SQSTM1 (step 5). Our earlier work [30] has shown that the Nt-Arg of R-HSPA5 binds the ZZ domain of SQSTM1 and allosterically activates the conformation of SQSTM1, exposing PB1 and LIR domains of SQSTM1 (step 6). This enables PB1-mediated self-aggregation of SQSTM1 along with R-HSPA5 and Ub-conjugated misfolded cargoes (step 7) and LIR-mediated interaction with LC3 (step 8), facilitating the autophagic removal of cytotoxic misfolded proteins and their aggregates. In this R-HSPA5-SQSTM1 circuit, PARK7 acts as a cofactor/co-chaperone of R-HSPA5 to modulate SQSTM1-dependent macroautophagy under TNFSF10-induced stresses and possibly other types of stress as well.