Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polγ against UV-induced inactivation

Abstract

Manganese superoxide dismutase is a nuclear encoded primary antioxidant enzyme localized exclusively in the mitochondrial matrix. Genotoxic agents, such as ultraviolet (UV) radiation, generates oxidative stress and cause mitochondrial DNA (mtDNA) damage. The mtDNA polymerase (Polγ), a major constituent of nucleoids, is responsible for the replication and repair of the mitochondrial genome. Recent studies suggest that the mitochondria contain fidelity proteins and MnSOD constitutes an integral part of the nucleoid complex. However, it is not known whether or how MnSOD participates in the mitochondrial repair processes. Using skin tissue from C57BL/6 mice exposed to UVB radiation, we demonstrate that MnSOD has a critical role in preventing mtDNA damage by protecting the function of Polγ. Quantitative-PCR analysis shows an increase in mtDNA damage after UVB exposure. Immunofluorescence and immunoblotting studies demonstrate p53 translocation to the mitochondria and interaction with Polγ after UVB exposure. The mtDNA immunoprecipitation assay with Polγ and p53 antibodies in p53(+/+) and p53(-/-) mice demonstrates an interaction between MnSOD, p53 and Polγ. The results suggest that these proteins form a complex for the repair of UVB-associated mtDNA damage. The data also demonstrate that UVB exposure injures the mtDNA D-loop in a p53-dependent manner. Using MnSOD-deficient mice we demonstrate that UVB-induced mtDNA damage is MnSOD dependent. Exposure to UVB results in nitration and inactivation of Polγ, which is prevented by addition of the MnSOD mimetic Mn(III)TE-2-PyP(5+). These results demonstrate for the first time that MnSOD is a fidelity protein that maintains the activity of Polγ by preventing UVB-induced nitration and inactivation of Polγ. The data also demonstrate that MnSOD has a role along with p53 to prevent mtDNA damage.

Figure 1. Quantification of mtDNA damage in mouse skin induced by UVB radiation

Wild-type C57BL/6 mice were exposed to 5kJ/m² of UVB radiation. mtDNA was isolated and analyzed with Q-PCR using primers specific for mouse mtDNA. The 10 kb mtDNA fragment was amplified and the blockage of rTth DNA polymerase amplification by damage in mtDNA was quantified in PCR products with Pico-Green dye. The relative amplification levels at 1 hr and 24 hr after UVB treatment were normalized with untreated control. ***P < 0.001 compared with control.

Figure 2. UVB enhances p53 mitochondrial translocation

(A) C57BL/6 mice were exposed to 5kJ/m² of UVB radiation. Whole skin tissue lysates and fractionated mitochondrial lysates were immunoblotted for p53 antibody (DO-1). Ponceau staining was used to confirm equal loading and uniform transfer of protein. Monoclonal anti-β-actin and anti-HSP60 antibodies were used as internal loading control. (B) The p53 accumulations in whole skin lysates and mitochondrial lysates at 1 hr and 24 hr after UVB exposure were quantified. Results were averaged from 3 sets of independent experiments. *P < 0.05, ***P < 0.001, compared with control, ##P < 0.01, comparison of 1 hr and 24 hr UVB treatment.

Figure 3. UVB-induced physical interaction of p53-Polγ-mtDNA

(A) Skin mitochondrial lysates from wild-type C57BL/6 mice exposed to 5kJ/m² of UVB radiation were immunoprecipitated with p53 (DO-1), Polγ antibodies, and control IgG. (B) mtDNA isolated from wild-type C57BL/6 mice skin exposed or sham exposed to 5 kJ/m² of UVB radiation was immunoprecipitated with p53 antibody (DO-1) and control IgG. The input mtDNA from control at 1 hr and 24 hr after UVB treatment was used as internal control. Real-Time PCR was used to amplify mtDNA D-loop region from input and immunoprecipitated mtDNA. ***P < 0.001. (C) mtDNA isolated from p53^+/+ and p53^−/− mice skin exposed or not exposed to 5kJ/m² of UVB radiation was immunoprecipitated with Polγ antibody. Real-time PCR was used to amplify the immunoprecipitated mtDNA D-loop. *P < 0.05, ***P < 0.001, compared with control; ##P < 0.01, compared at 1 hr and 24 hr after UVB treatment; ^P<0.05, compared between p53^+/+ and p53^−/− genotypes.

Figure 4. UVB-induced physical interaction of MnSOD-p53-Polγ

(A) Skin mitochondrial lysates from wild-type C57BL/6 mice exposed to 5kJ/m² of UVB radiation were immunoprecipitated with p53 (DO-1), MnSOD antibodies, and control IgG. Co-immunoprecipitated MnSOD and p53 were quantified by immunoblotting with specific antibodies. (B) Co-immunoprecipitation of Polγ and MnSOD. Mitochondrial lysates from mice skin exposed to UVB at 1 hr and 24 hr were immunoprecipitated with Polγ, MnSOD antibodies, and control IgG. Co-immunoprecipitated Polγ and MnSOD were quantified by immunoblotting with specific antibodies. (C) Mitochondrial lysates from p53^+/+ and p53^−/− mice skin exposed to UVB were immunoprecipitated with Polγ, MnSOD antibodies and control IgG. Co-immunoprecipitated Polγ and MnSOD were quantified by immunoblotting with specific antibodies. (D) mtDNA isolated from MnSOD^+/+ and MnSOD^+/− mice skin exposed to 5kJ/m² of UVB radiation was immunoprecipitated with Polγ antibody. Real-time PCR was used to amplify the precipitated mtDNA D-loop. ***P < 0.001, **P < 0.01, compared with control; ^^P < 0.01 compared at 1 hr and 24 hr after UVB treatment; ##P < 0.01 compared between MnSOD^+/+ and MnSOD^+/− mice.

Figure 5. UVB induced Polγ inactivation by nitration

(A) Immunoblot analysis of Polγ protein levels in MnSOD^+/+ and MnSOD^+/− mice skin mitochondria exposed to 5kJ/m² of UVB radiation. (B) Polγ reverse transcriptase activity assay in MnSOD^+/+ and MnSOD^+/− mice skin mitochondria exposed or not exposed to 5 kJ/m² of UVB radiation #P < 0.05, ###P < 0.001 compared with control. (C) MnSOD^+/+ and MnSOD^+/− mouse skin exposed to 5 kJ/m² of UVB radiation was co-immunoprecipitated with 3-nitrotyrosine antibody. Co-immunoprecipitates were immunoblotted with Polγ antibody. (D) Quantification of Polγ co-immunoprecipitation by 3-nitrotyrosine antibody. **P < 0.01, ***P<0.001 compared with control; #P < 0.05 compared between 1 hr and 24 hr after UVB treatment; ^P < 0.05 compared between MnSOD^+/+ and MnSOD^+/− mice.

Figure 6. Mn^IIITE-2-PyP⁵⁺ protects UVB induced Polγ inactivation by nitration in MnSOD^+/− mice

(A) Polγ reverse transcriptase activity assay in Mn^IIITE-2-PyP⁵⁺ and saline pre-treated MnSOD^+/− mice skin mitochondria exposed to 5 kJ/m² of UVB radiation *P < 0.05 compared with control; #P < 0.05 compared between 1 hr and 24 hr after UVB treatment; ^P < 0.05 compared between Mn^IIITE-2-PyP⁵⁺ and saline pre-treatment. (B) Mn^IIITE-2-PyP⁵⁺ and saline pre-treated MnSOD^+/− mice skin exposed to 5 kJ/m² of UVB radiation were co-immunoprecipitated with 3-nitrotyrosine antibody and immunoblotted with Polγ antibody. (C) Quantification of Polγ co-immunoprecipitation by anti-3-nitrotyrosine antibody in Mn^IIITE-2-PyP⁵⁺ and saline pre-treated MnSOD^+/− mice skin exposed to 5 kJ/m² of UVB radiation. **P < 0.01, ***P < 0.001 compared with control; ###P < 0.001 compared between 1 hr and 24 hr after UVB treatment; ^^P < 0.01 compared between Mn^IIITE-2-PyP⁵⁺ and saline pre-treatment.

Figure 7

Schematic illustration of novel dual-step strategy adapted by keratinocytes to survive UVB insult