Fig 1: KEAP1 controls the degradation of full-length PGAM5. A. KEAP1 interacts only with full-length PGAM5. The left panel shows that full-length PGAM5-FLAG, but not cleaved PGAM5, co-immunoprecipitates with KEAP1-HA in HEK cells. Note also that PGAM5 E79A/S80A co-immunoprecipitates with KEAP1 less efficiently than PGAM5-WT-FLAG. The right panel shows quantification of full length and cleaved PGAM5-WT from total cell lysate and KEAP1 interacting fraction. ****P < 0.0001 compared with respective total cell lysate group, n = 3 independent IP-s, one-way ANOVA followed by Sidak's multiple comparison test. B. KEAP1 overexpression induces degradation of PGAM5. The left panel shows the representative Western blot image of PGAM5 expression in the presence of KEAP1-FLAG or KEAP1 R380A/R415A-FLAG in HEK cells. Note also that KEAP1-FLAG but not KEAP1 R380A/R415A-FLAG is co-immunoprecipitating with PGAM5-HA. The right panel shows quantification of full length and cleaved PGAM5 from total cell lysate. **P < 0.01, ns: not significant, n = 4 independent experiments, one-way ANOVA followed by Sidak's multiple comparison test. C. KEAP1 silencing increases the level of endogenous PGAM5. Representative Western blot image (left) and analysis of PGAM5 expression (right) in PC6 cells expressing scrambled or KEAP1 shRNA. ***P < 0.001 and ****P < 0.0001, n = 4, t-test. D. PGAM5 is accumulating in the cytosol after proteasome inhibition. Representative superresolution Airyscan images showing PC6 cells expressing PGAM5-YPet and mitochondrially targeted Kate2 treated with DMSO or 25 µM MG132 for 5 h. E. Proteasome inhibition leads to cytosolic accumulation of full-length PGAM5. Representative Western blot image of PGAM5-Flag levels in cytosolic and mitochondrial fractions of PC6 cells treated DMSO or 25 µM MG132 for 5 h. ATP5A and GAPDH as markers of mitochondria and cytosol, respectively, demonstrate the purity of fractions.
Fig 2: Inhibitors of KEAP1–PGAM5 protein-protein interaction enhance mitophagy. A. Molecular docking simulation showing energetically most favorable interactions between a KEAP1 and non-electrophilic KEAP1 inhibitors. The table is showing hydrogen-bond pairings in KEAP1-inhibitor complexes. The length of the hydrogen bond is given in angstroms. B. Treatment with CPUY192018 disrupts KEAP1-PGAM5 interaction. PC6 cells were co-transfected with KEAP1-FLAG and PGAM5-HA and treated for 24 h with DMSO or 100 µM CPUY192018. KEAP1-FLAG was co-immunoprecipitating with PGAM5-HA in DMSO but not in CPUY192018 treated cells. C. Treatment with CPUY192018 increases the level of endogenous PGAM5. PC6 cells were treated with 100 µM CPUY192018 for 24 h. The left panel shows a representative Western blot and the right panel quantitative analysis. *P < 0.05, n = 4 samples, t-test. D. Effect of non-electrophilic KEAP1 inhibitors on mitochondrial membrane potential. PC6 cells were treated with KEAP1 inhibitors for 24 h, after which the cells were stained with ratiometric mitochondrial membrane potential sensor JC10. *P < 0.05 and ****P < 0.0001 when compared with DMSO treated group, n = 7–12 wells per data point, One-way ANOVA followed by Dunn's multiple comparisons test. E. Non-electrophilic KEAP1 inhibitors induce Parkin-EYFP translocation to mitochondria. PC6 cells expressing Parkin-EYFP were treated with DMSO or 100 µM of KEAP1 inhibitors for 24 h ***P < 0.001 and ****P < 0.0001 when compared with DMSO treated group, n = 6 dishes per group, 20 fields per dish, One-way ANOVA followed by Dunnett's multiple comparisons test. F and G. Non-electrophilic KEAP1 inhibitors enhance mitophagy but not general autophagy. Neurons expressing mitochondrial marker Kate2 and autophagosome markers EGFP-LC3B and GFP-LC3C were treated with DMSO or 100 µM of KEAP1 inhibitors for 24 h. The number of LC3 positive puncta colocalizing with mitochondria (F) as well as the total number of LC3 positive puncta (G) was counted by a blinded observer. **P < 0.01, ***P < 0.001 and ****P < 0.0001 when compared with DMSO treated group, n = 9–14 dishes (10 cells per dish), Welch's ANOVA followed by Dunnett's T3 multiple comparisons test. H. Treatment of primary cortical neurons with CPUY192018 (24 h) induces a concentration-dependent increase in mitochondrial colocalization with autophagosome markers EGFP-LC3B/GFP-LC3C. *P < 0.05 and ****P < 0.0001, when compared with DMSO, treated group, n = 16–18 dishes from 4 independent experiments (10 cells per dish), Kruskal-Wallis test followed by Dunn's multiple comparisons test, the dotted line shows the EC50.
Fig 3: Immunofluorescence staining of peripheral blood leukocytes (PBLs): (a) the expression of KEAP1; (b) the expression of NRF2. The fluorescence localization of KEAP1 and NRF2 was measured with immunofluorescence. Anti-NRF2 and KEAP1 antibodies were used to detect NRF2 and KEAP1 localization (green) using a fluorescence microscope. DAPI staining indicated the locations of the nuclei (blue). n = 10 in each group, and intervention duration was 3 months. (a) KEAP1 expression in the CG group. (b) KEAP1 expression in the IG group. (c) NRF2 expression in the CG group. (d) NRF2 expression in the IG group.
Fig 4: Effect of KEAP1 knockdown on H2O2-induced alterations in Hep2 cell apoptosis. (A) H2O2 evidently induced cell apoptosis from 14.1 to 27.9 and 31.8 to 45.3% in shKEAP1 and scHep2 cells, respectively. (B) The apoptotic rate in shKEAP1 Hep2 cells was lower compared with in scHep2 cells. *P<0.05 vs. the shKEAP1 at the corresponding H2O2 concentration. KEAP1, kelch-like ECH-associated protein 1; H2O2, hydrogen peroxide; H2O2, hydrogen peroxide; sh, short hairpin RNA; scHep2, scrambled control-transfected Hep2 cells; APC, allophycocyanin; 7-AAD, 7-aminoactinomycin D. The lower right-hand side quadrant was considered to indicate apoptotic cells.
Fig 5: CORM-A1 improves oxidative stress and cell survival via Nrf2 translocation and activation of ARE genes in hepatocytes. HepG2 cells were treated with PA (100 µm) in presence and absence of CORM-A1 (100 µm). Oxidative stress in HepG2 cells was evaluated using (A) CellROX and DHE stain in control, PA, PA + CORM-A1 and CORM-A1 groups. Cell survival was determine using (B) cell viability assay (MTT) (C) Nrf2 translocation was checked using immunoblot analysis in cytoplasm and nucleus (D) co-immunoprecipitation demonstrating time depended decrement in keap1 protein (E) qualitative and quantitative levels of HO-1 and Keap1 proteins and (F) expression levels of antioxidant gene was recorded using qPCR analysis. Results expressed as mean ± S.E.M. *p < 0.05, **p < 0.01 or ***p < 0.001 is when PA treatment is compared to control cells and PA + CORM-A1 is compared to PA alone group.
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