| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 96 === Zac1 (Zinc–finger protein which regulates apoptosis and cell cycle arrest 1) acts as a transcriptional cofactor for a number of nuclear receptors and p53 and, like other transcriptional factors and cofactors, its function should be modulated by post-translation modification. The interesting issue in this thesis was to study whether the sumoylation modification for the regulation of transcriptional activation and coactivation of Zac1 because of the physical and functional interactions between Zac1 and Ubc9, which is the only one E2 enzyme for the sumoylation pathway. Sumoylation modifies protein function via the formation of a covalent bond by small ubiquitin-like modifier-1 and can act as a transcriptional repressor. Interestingly, Dominant-negative Ubc9 (C93S), which is lack of the E2-conjugating enzyme activity, still positively regulates some transcriptional activations. This thesis found that wild-type and dominant-negative Ubc9 were able to enhance the transactivation activity of CBP and its mutant lacking sumoylation sites, Zac1 further enhanced the activity through protein-protein interaction with CBP and Ubc9 independent of the sumoylation modification in HeLa cell. Daxx (death domain-association protein) selectively modulated Zac1-induced CBP transactivation activity and endogenous p53 protein might be required for its effects. With the help of computer program, there are three potential SUMO modification sites at lysine residues 36, 237, 424 of Zac1. Three point mutants of these amino acids expressed higher transactivation activity than wild-type Zac1. Wild-type and mutant Zac1 expressed similar roles on AR- and p53-dependent transcriptional activations and Daxx and its SIM-defective mutant had the ability to negatively regulate Zac1-induce AR- or p53-dependent transactivation activities, indicating that there have sumoylation dependent and independent pathways. However, the detailed mechanisms have to be more identified.
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