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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物醫學研究所 > 依資料類型分類 > 碩博士論文 >  182位置酪胺酸的磷酸化降低Rad23蛋白與泛素的結合能力及其所參與的蛋白質降解途徑

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/100947

標題: 182位置酪胺酸的磷酸化降低Rad23蛋白與泛素的結合能力及其所參與的蛋白質降解途徑
Phosphorylation of Tyrosine-182 decreases the ubiquitin binding ability and Rad23-mediated protein degradation
作者: 陳柏蓉
Bo-RongChen
Contributors: 闕斌如;林芸薇
莊秀美
中興大學
關鍵字: Rad23;phosphorylation
磷酸化
日期: 2009
Issue Date: 2012-09-04 10:49:36 (UTC+8)
Publisher: 生物醫學研究所
摘要: Rad23為一個從酵母菌到人類間具高度保留性的蛋白,其參與於細胞內的核甘酸修補機制及泛素系統所主導的蛋白質降解途徑;然而,目前細胞是透過何種機制調控Rad23參與於這些功能仍然未知。為了研究細胞內訊號對Rad23功能的調控,利用定位點突變技術及體外激酉每試驗證明182位置的酪胺酸在物種間具高度保留性且可以被激酉每Rad53磷酸化,Y182F/D的突變不影響Rad23蛋白自身的穩定度。在功能的影響分析上發現Y182F/D的突變株不影響細胞對UV的敏感性及Rad23與Rad4的結合能力;但Y182D的突變顯著地降低了Rad23與K48及K63連接型的聚泛素鏈的結合力及些微地降低了Rad23與26S蛋白水解酉每複合體間的結合。而經由UFD途徑降解的Ub-Pro-β-gal,在高度表現Rad23及Y182F的突變株中的穩定度大為增加,但在表現Y182D的突變株中則觀察不到此情形。此外,Y182D的突變降低了Rad23形成同型雙體的能力。基於觀察到的結果,推測182位置酪胺酸的磷酸化降低Rad23蛋白與泛素的結合能力及其所參與的蛋白質降解途徑;但其作用的分子機轉仍需進一步的研究探討。
Rad23 is evolutionarily conserved for involving in DNA nuclear excision repair (NER) and ubiquitin-proteasome system (UPS) from yeast to human. However, the signaling regulation of Rad23 function is still unknown. To investigate the signaling control for Rad23 function, site-directed mutagenesis and in vitro kinase assay were used to prove Tyrosine-182, the highly conserved residue in UBA1 domain among species, is phosphotylated by Rad53. Y182F/D mutants didn't affect the stability of Rad23 itself. Functional analysis showed that Y182F/D mutants were similar to wild-type in UV sensitivity and Rad4 binding affinity. Interestingly, Y182D mutant significantly reduced the K48- and K63-linked polyubiquitin chain binding ability in vitro and in vivo and slightly decreased the association with 26S subunits. Ub-Pro-βgal, a substrate of the ubiquitin-fusion degradation pathway, was significantly stabilized in strains that over-expressed wild-type Rad23 and Rad23Y182F, but not Y182D mutant. Furthermore, the lower dimerization was observed in wild-type /Rad23Y182D and Rad23Y182D/ Rad23Y182D. Based on these results, we suggest that the ubiquitin chain binding ability of Rad23 might be down-regulated through the phosphorylation of Y182 on UBA1 domain, which consequently affect the Rad23-mediated proteolysis. However, the molecular mechanism for Y182 phosphorylation by signaling will be further investigated.
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