The Research Of Modification On Human TAF41 Protein By SUMO

As a nuclear protein, TAF41 (TBP-associated factors 41) has been demonstrated to function in RNA polymeraseI transcription and interact with DNA Polymeraseβ. TAF41 is highly expressed in human carcinomas and tumor cell lines but not in normal tissues, suggesting it is most likely involved in carcinogenesis. There is experimental evidence that the TAF41 involved in the occurrence of ovarian cancer, but its function and mechanism is not entirely clear.SUMO (small ubiquitin-like modifier) is a recently discovered post-translational modifier. It has remarkable similarity with ubiquitin both on protein secondary structure and modification process. However unlike ubiquitination, which primarily targets substrate proteins to the proteasome for degradation, SUMOylation play important role in a number of cellular processes such as transcriptional regulation, nuclear transport, cell cycle modulation, apoptosis and DNA repair. SUMO modulates protein particular function though covalently modify to the lysine residue of target protein.Here we found out that TAF41 protein may contain two potential SUMOylation sites by analyzing its protein sequence. It can be SUMOylated on either 42 lysine residue or 168 lysine residue. This suggested that TAF41 maybe a new substrate protein of SUMOylation, however, how TAF41 modified and how its modifications influence the transcription pathway is still unreported. In this study, we first construct eukaryotic expression plasmids encoding WT-TAF41. With pCMV-HA/Myc-TAF41 as a template, the mutant plasmids K42R, K168R, K42/168R were obtained by using site-directed mutagenesis technology. The recombinant plasmid and all mutants were confirmed though the analysis of enzyme digestion and DNA sequencing. In order to determine protein-protein interaction of TAF41 and SUMO-1, TAF41 recombinant plasmids and its mutants were respectively co-transfected with SUMO-1 into 293 cells by lipofectin method. And then Immunofluorescence was used to observed sub-cellular distribution of TAF41 and colocalization of SUMO-1 and TAF41. Immunoprecitation and Western bolt protocol was then used to demonstrate TAF41 Sumoylation, and which mutant site is specific Sumoylation site. The results suggest that TAF41 is a substrate of SUMOylation, and that 168 lysine residue is the major SUMOylation site of TAF41, and that modification of TAF41 by SUMO may modulate the transcriptional repression effect of TAF41 on the p53 transcription pathway.