Hiv-1 Tat Protein Of Dna-pkcs

Several types of cancers have been considered to be strongly associated with the HIV-1 infection and AIDS. These are Kaposi sarcoma (KS), lymphoproliferative disorders and cervical cancer, etc. Tat is a HIV encoded small protein secreted early after infection and is absolutely required for efficient transcription of HIV-1 provirus and viral replication. Tat is secreted by HIV-infected cells, and taken up by and affects neighbouring cells. This"bystander"effect is able to affect both infected and uninfected cells, therefore is regarded as an important contributory to the pathogenesis of AIDS.Cells have developed many mechanisms to protect the integrity of genomic DNA and maintain genomic stability. These mechanisms are important for cell and organism survival and normal growth control. Surveillance protein complexes monitor the integrity of the genome. DNA damage and chromosomal structure aberrations such as double-strand DNA breaks coordinately trigger the cell cycle checkpoint pathways and DNA repair systems. Activation of a DNA damage checkpoint results in cell cycle arrest, allowing time for DNA repair. Cells possess many DNA repair systems, which can recognize and repair specific types of DNA damages. The DNA double-strand break (DSB) is the most harmful form of DNA damages. In order to combat this potentially lethal DNA lesion, cells have developed two major mechanistic pathways to repair DSBs, i.e. the homologous recombination (HR) and non-homologous DNA end-joining (NHEJ). DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex, in which there are another two regulatory components, Ku70 and Ku80. DNA-PKcs is a serine/threonine protein kinase that belongs to the members of the (PIKK) superfamily which includes ATM, ATR and so on.We utilized the purified Tat protein and the cellular model of expressing exogenous HIV-1 tat gene in a human rhabdomyosarcoma cell line, to investigate the effects of Tat protein on cellular DNA damage response and its related mechanism, especially regarding the effect on function of DNA-PKcs. The results and conclusions were summarized as following:(1) Tat protein was efficiently expressed in E.coil of Rosetta-gami B(DE3), and we got the Tat protein (purity>95%) purified via affinity chromatography. The transcriptional activation function of Tat was confirmed by utilizing Tat transcriptional activating reporter vector KB/SP1LTR—nlacZ. The exogenous Tat protein can efficiently enter into the cell nuclei and its half-time in the cell is about 4h.(2) Tat protein was found to suppress the transcriptional activity of DNA-PKcs gene promoter via the Dual-Luciferase Reporter Assay System. The Tat protein not only inhibits the activity of DNA-PKcs gene core promoter, but also inhibits the rest elements beyond the core's sequence. We identified that Tat protein can't directly bind the DNA-PKcs'core promoter by means of EMSA. We confirmed that Tat has the effect of large scale chromatin relax (remodeling). This chromatin remodeling effect of tat protein was deminshied when the cells exposed to ionizing radiation. Evidence that tat acetylation is important for its chromatin remodeling function comes from the treatment with acetylase inhibitor anacardic acid (AA) and the point-mutation experiments, in which either K28 ,K50 or K51 mutation could decrease its ability of chromatin relax.(3) Autophosphorylation of DNA-PKcs at Ser-2056 was found to be up-regulated by Tat. The phosphorylated DNA-PKcs at Ser-2056 was significantly increased in TE671tat cells at different time after IR as as compared to parental cells. Using an in vitro kinase assay, we clearly showed that in the presence of Tat, DNA-PKcs displayed an increased kinase activity. We confirmed the interaction between Tat and DNA-PKcs in vivo and vitro via using co-immunoprecipitation and GST-pull down. The colocalization of Tat and DNA-PKcs in the nulear of HeLa cells was also observed by immunofluorence technology. The interaction and binding domain of DNA-PKcs with Tat were determined via co- immunoprecipitation. FAT domain of DNA-PKcs was idenfied to be Tat binding domain. Furthermore, the interaction of Ku70, Ku80 and Tat was defined by co- immunoprecipitation.(4) Immunofluorescence experiments using confocal microscopy showed that obviousγ-H2AX foci were induced in the cell nuclei by Tat. The comet assay showed that exposure of Tat resulted in DSB in TE671. A flow-cytometry and fluorescence microscope analyses revealed that an inceased level of ROS in TE671tat cells as compared with the control cells. Accordingly, the level of MDA was higher and the expression of GSH was even lower in the Tat protein treated cells as compared with that in control cells. Western blotting anaysis showed that Tat could suppress the protein expression of Prx1,2,5,6 and Cu/ZnSOD. (5) A G2/M arrest was induced in parental 293T cells at as early as 4hr after 4Gy irradiation. However, 293T cells in the presence of Tat exhibited a noticeable delay on the initiation and elimination of radiation-induced G2/M arrest and a prolonged S phase arrest as compared to parental cells.(6) We confirmed the interaction between Tat and Plk1 as well as Chk2 in vivo and vitro via using co-immunoprecipitation and GST-pull down. By cotransfecting the RFP-tat and GFP-Plk1 into 293T or HeLa cells, they were colocalized in the nuclear. The amino-terminal kinase domain of Plk1 was identified as the the interaction domain with Tat by GST-pull down assay. Phosphorylation of Chk2 at Thr-68 was found to be up-regulated by Tat, the level of phosphorylated Chk2/pT68 was significantly increased in TE671tat cells at different time after ionizing radiation as compared with parental TE671 cells.