| Objective :Deinococcus radiodurans(DR)is a microorganisms which exhibits a dramatic ability to withstand the lethal and mutagenic effects of ionizing radiation and ultraviolet radiation. Such an extraordinary resistance to radiation is closely related to its perfect and highly efficient DNA repair system. PprI is a unique regulatory protein in Deinococcus radiodurans which plays an important role in regulation of DNA damage repair and is considered to be the main switch of DNA damage repair pathway in Deinococcus radiodurans. Since the discovery of more than 50 years of Deinococcus radiodurans, the effects and mechanisms against radiation of PprI protein in prokaryotes has been deeply studied. In this work, we investigate the expression and purification methods of PprI protein and TAT-PprI protein in eukaryotic system,and its effects and mechanisms against radiation in eukaryotic cells in order to provide valuable experimental data for clinical application of acute radiation injury. So far has not been reported in the literature at home and abroad.Materials and Methods: In this work, we used pHBM905A-His-PprI vector constructed by Wu Wei and pPICZα A-TAT-PprI vector constructed by Qiao Huiping as templates to optimize the expression and purification conditions in Pichia pastoris in order to obtain a large number of PprI protein and TAT-PprI protein from Deinococcus radiodurans. Human umbilical vein endothelial cells(HUVEC) were used as the research object. The distribution of PprI protein and TAT-PprI protein inHUVECs was observed by immunofluorescence experiments. The influence on the cell toxicity and cell proliferation after irradiation about the proteins were detected by CCK-8 and the influence on cell survical after irradiation was detected by clone formation experiment. For the other hand, intracellular ROS levels in irradiated HUVECs were detected by the fluorescent probe DCFH-DA meanwhile the activity of the antioxidant proteins superoxide dismutase(SOD) and catalase(CAT) and the concentrations of malondialdehyde(MDA) within irradiated HUVECs were studied by antioxidant kits. The influence on the cell apoptosis rates and the expression of mitochondrial apoptosis pathway related proteins(Bcl-2、Bax) about PprI protein and TAT-PprI protein were detected respectively by flow cytometry and immunoblotting.Additionally, the influence on γH2AX foci formation and the expression of eukaryotic recombination repair protein(Rad 51) about PprI protein and TAT-PprI protein were detected respectively by immunofluorescence experiments and immunoblotting.Results:1. The expression and purification conditions of PprI protein and TAT-PprI protein were successfully optimized,and 5 mg PprI protein and 5 mg TAT-PprI protein were efficiently obtained at once.2. Compared with PprI protein, TAT-PprI protein has a strong trans-membrane ability, and acted on organelles and nucleus in cells.3. The results of cell toxicity test indicted that PprI protein and TAT-PprI protein from Deinococcus radiodurans in the concentration range of 0-10 and 0-25 μg/ml respectively were nontoxic to the cells(P>0.05). When the PprI protein concentration was greater than 20 μg/ml, it has inhibitory effect on the growth of cells(P<0.05)meanwhile when the function time reached to 24, 48, 72 h, the half maximal inhibitory concentration(IC50) was 63.58, 51.80 and 32.46 μg/ml respectively. When the TAT-PprI protein concentration was greater than 50 μg/ml, it has inhibitory effect on the growth of cells(P<0.05) meanwhile when the function time reached to 24, 48, 72 h,the IC50 was 107.90、97.26、87.55 μg/ml respectively.4. The results of cell proliferation experiment showed that PprI protein andTAT-PprI protein in the concentration range of 0.8-8 μg/ml had obvious effect on promoting proliferation of the irradiated cells(P<0.05~0.001).5. Cell survival curve indicted pretreatment with PprI protein and TAT-PprI protein resulted in a significant increase of HUVEC viability at 2, 4, 6 and 8 Gy levels of irradiation and a higher survival curve parameter of D0, Dq, N and SF2 as compared to irradiation group(P<0.05~0.001). Additionally, pretreatment with TAT-PprI protein resulted in a significant increase of HUVEC viability at both 4, 6 and 8 Gy levels of irradiation compared to PprI protein(P<0.05).6. The results of antioxidant experiments showed that PprI protein and TAT-PprI protein significantly reduce ROS levels(P<0.001) and MDA concentrations(P<0.05).On the other hand, pretreatment with TAT-PprI protein significantly increased levels of SOD and CAT activity(P<0.05) compared to irradiation group while pretreatment with PprI protein showed no significant change(P>0.05).7. The results of cell apoptosis experiments showed pretreatment with PprI protein and TAT-PprI protein significantly reduced the basal apoptosis rate compared to irradiation group(P<0.01~0.001), and pretreatment with TAT-PprI protein significantly increased expression of Bcl-2 and reduced the expression of Bax(P<0.01~0.001) while pretreatment with PprI protein showed no significant change in the expression of Bcl-2 and Bax(P>0.05).8. The results of DNA damage repair experiments showed that pretreatment with PprI protein and TAT-PprI protein markedly reduced numbers of γH2AX foci after irradiation(P<0.05) and exhibited radically increased levels of Rad51 protein expression compared to the control(P<0.01~0.001).Conclusion:1. We successfully optimized the expression and purification conditions of Pichia pastoris recombinant pHBM905A-His-PprI/GS115 strain and pPICZα A-TAT-PprI/X33 strain which contain the pprI gene of Deinococcus radiodurans, and obtained a large number of PprI protein and TAT-PprI protein efficiently.2. TAT protein transduction domain can mediate PprI protein across cellularmembranes and widely distributes in the cytoplasm and nucleus.3. The results of cytotoxic assay indicted that 0-10 μg/ml of PprI protein and 0-25μg/ml of TAT-PprI protein were nontoxic to the cells. These proteins have a wide safe dosage window.4. The results of cell proliferation experiment showed that among the safe dose screened out above, PprI protein and TAT-PprI protein had a wide dosage window(0.8-8 μg/ml) in which had obvious effect of promoting proliferation on the irradiated cells.5. PprI protein and TAT-PprI protein increased median lethal dose, enhanced radioresistance of cells and repair capacity of radiation damage. Additionally,TAT-PprI protein had more significant anti-radiation effects.6. PprI protein and TAT-PprI protein significantly reduced intracellular ROS levels, enhanced the activities of antioxidant enzymes and alleviated the degrees of cell lipid peroxidation. Compared with PprI protein, TAT-PprI protein had more significant antioxidant effect.7. PprI protein and TAT-PprI protein significantly reduced the apoptosis rates of cells after irradiation and compared with PprI protein, TAT-PprI protein played a more significant role in regulating the expression of apoptosis mitochondrial channel proteins.8. PprI protein and TAT-PprI protein markedly reduced numbers of γH2AX foci after irradiation and exhibited radically increased levels of Rad51 protein expression.Compared with PprI protein, TAT-PprI protein had more significant DNA repair effect.In conclusion, we obtained highly efficient expression and purification of PprI and TAT-PprI protein from Deinococcus radiodurans in Pichia pastoris for the first time whether at home or abroad, and successfully used in prevention of acute radiation injury of human cells. It was found that PprI and TAT-PprI protein have a significant effects of anti-radiation damage, and the effects were closely related to the mechanisms of action on anti-radiation, anti-apoptosis and enhancement of DNA damage repair in the cells. Furthermore, anti-radiation effect of TAT-PprI protein was significantsuperior to that of PprI protein due to its ability to across cytomembrane. |
PDF Full Text Request