HIV-TAT Mediated Protein Transduction Of Cu/Zn-superoxide Dismutase-1（SOD₁） Protects Skin Cells From Ionizing Radiation

Part ⅠConstruction of TAT-SOD1prokaryotic expression vector, expression andpurification of recombinant TAT-SOD1proteinObjectives: The commonly used skin protective sprays against ionizing radiationconsists of superoxide dismutase (SOD), which can catalyze the dismutation ofsuperoxide (O2·) into oxygen and hydrogen peroxide. SOD1protein was unable to crossthe cell membrane into the cells due to the large molecular weight, which limits itsapplication. Noreover, SOD derived from animal blood remains a serious problem. Thefusion of protein transduction domain (PTD) HIV-TAT and human copper-zincsuperoxide dismutase (Gu/Zn-SOD, SOD1) could overcame this difficulty. ThisTAT-SOD1fusion protein was expressed and purified in E. Coli and he membranetransduction effect and biological effect of TAT-SOD1was verified.Methods: The coding sequence of the HIV-TAT domain (YGRKKRRQRRR) wassynthesized and subcloned into the BamHI and EcoRI sites of pET-28a. Human SOD1cDNA was amplified by PCR using appropriate primers and then subcloned into theEcoRI and XhoI sites of pET-28a-TAT to generate pET-28a-TAT-SOD1. The controlvector, which expressed SOD1, was constructed by inserting SOD1coding sequence intopET-28a without TAT domain. The expression of recombinant protein was in the host E.coli BL21(DE3). Cells were sonicated, the columns of Ni-nitrilotriacetic acid agarosewere used to purify protein. Immunofluorescence assay was used to verify thetransmembrane roles of TAT-SOD1. SOD activity assay, superoxide anion scavengingassay and total antioxidant capacity assay was used to measure the biological activity ofTAT-SOD1protein intracellular.Results：The pET-28a-TAT-SOD1prokaryotic expression vector was successfullyconstructed. After transformed into E.coli, high purity recombinant TAT-SOD1protein was obtained by inducible expression and purification. Immunofluorescence assayshowed that the recombinant protein was able to cross the cell membrane into the cell.SOD activity assay, superoxide anion scavenging assay and total antioxidant capacitymeasured test results indicated that the recombinant protein retained biological activity.Conclusions：Prokaryotic expression vector pET-28a-TAT-SOD1was transformedinto E. coli, the recombinant TAT-SOD1protein was expressed and purified. Uptake ofTAT-SOD1by HaCaT cells retained its biological activity. Part ⅡHIV-TAT mediated protein transduction of Cu/Zn-superoxide dismutase-1(SOD1) protects skin cells from ionizing radiationObjectives: The aim of this study was to evaluate the protective role of HIV-TATprotein transduction domain mediated protein transduction of SOD1(TAT-SOD1)against ionizing radiation in human keratinocyte-derived HaCaT cells and SD ratmodels..Methods: The cell viability was detected by CCK-8assay. Annexin V-PI doublestaining and Western Blot was conducted for the detection of cell apoptosis. TheDCFH2-DA assay was used to detect the free radicals induced by ionizing radiation.Mito-Tracker staining assay was used to reflect ionizing radiation-inducedmitochondrial damage, DNA double-strand breaks (DSB) were detected byimmunofluorescence detection of γH2AX. After irradiated by45Gy electron beams, thelipid peroxidation and free radical levels of SD rats skin was detected byMalondialdehyde (MDA) and DCFH2-DA assay.Results：Compared with natural SOD1, the application of TAT-SOD1significantlyenhanced the viability and decreased the apoptosis induced by X-ray irradiation.Moreover, TAT-SOD1reduced ROS and preserved mitochondrial integrity afterradiation exposure in HaCaT cells. Radiation-induced γH2AX foci, which arerepresentative of DNA double strand breaks, were decreased by pretreatment withTAT-SOD1. Furthermore, subcutaneous application of TAT-SOD1resulted in asignificant decrease in45Gy electron beam-induced ROS and MDA concentration inthe skins of rats. Conclusions：This study provides evidences for the protective role of TAT-SOD1inalleviating radiation-induced damage in HaCaT cells and rat skins, which suggests anew therapeutic strategy for radiation-induced skin injury.