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標題: 酵母菌Rad23蛋白47位置絲氨酸的磷酸化降低與26S 蛋白酶體的結合能力及其所參與的蛋白質降解途徑
Phosphorylation of Serine 47 on Rad23 protein reduces the 26S proteasome binding ability and Rad23-mediated protein degradation in Saccharomyces cerevisiae
作者: 柯博庭
Ko, Bo-Ting
關鍵字: Rad23
protein degradation
出版社: 生物醫學研究所
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摘要: Rad23為一個從酵母菌到人類間具有高度演化保留性的蛋白,研究證實Rad23參與細胞內的核苷酸修補機制及泛素系統所主導的蛋白質降解途徑並且扮演著重要的角色。然而,訊號傳遞機制如何調控Rad23參與這些系統仍舊未知。Rad23 UBL區塊可以被Rad53激酶磷酸化,以定位點突變技術及活體外激酶試驗證實Rad23蛋白47位置絲胺酸為Rad53磷酸化位置。S47位置磷酸化與否並不影響與Rad4之間的交互作用及Rad4蛋白的穩定性。有趣的是,S47的磷酸化降低酵母菌對UVC照射的存活率。活體內及活體外結合試驗則證實Rad23磷酸化顯著的降低與26S蛋白酶體的結合能力。另一方面,經由UFD途徑降解的Ub-Arg- and Ub-Pro-β-gal,在表現S47A的突變株中穩定性大為增加。另一方面,生理功能試驗則證實了UBA或UBL區塊的突變導致細胞喪失對不同的生存壓力的對抗能力。基於上述的結果,推測UBL區塊47位置絲胺酸的磷酸化降低Rad23蛋白與及26S蛋白酶體的結合能力及其所參與的蛋白質降解途徑。然而,訊號機制如何調控47位置絲胺酸的磷酸化仍須進一步的探討。
Rad23 is an evolutionarily conserved protein from yeast to human. Researches have demonstrated that Rad23 played an important role in both nuclear excision repair (NER) and ubiquitin-proteasome system (UPS) mediated protein degradation pathway. However, well-defined mechanisms control Rad23 in these systems still unavailable. Rad23 could be phosphorylated by Rad53 on UBL in vitro. Site-directed mutagenesis and in vitro kinase assay proved that Serine-47 could be phosphorylated by Rad53. S47A/E mutants do not affect the interaction of Rad4 and Rad4 protein stability. Interestingly, Serine-47 phosphorylation decreases survival rate under UVC irradiation. Besides, in vivo and in vitro binding assay showed that Rad23S47E significantly decreased the association with 26S subunits. Ub-Arg- and Ub-Pro-β-gal, the substrates of the ubiquitin-fusion degradation pathway, were significantly stabilized in cells expressing wild-type Rad23 and Rad23S47A, but not S47E mutant. In addition, biofunctional assay demonstrated that mutations in UBL or UBA domain reduced cells ability to against different survival stress. Based on these results, we suggest that the 26S proteasome binding ability and UPS-mediated proteolysis might be down-regulated by Serine-47 phosphorylation. However, the precisely molecular mechanisms control S47 phosphorylation should be further investigated.
其他識別: U0005-2907201016392300
Appears in Collections:生物醫學研究所



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