大腸桿菌ClpY 對基質SulA專一辨識位之研究

Abstract

ATP依賴蛋白酶普遍存在於細菌、古菌以及真核細胞中，這一類型的蛋白酶主要的功能為調節其他蛋白質在細胞中的量，分解不正常堆積的蛋白質或將折疊錯誤之蛋白質調整回正常構型。ClpYQ/HslUV蛋白酶為ATP依賴蛋白酶其中的ㄧ種，是由ClpY(50 KDa)和ClpQ(19 KDa)構成，各自形成六元環後複合成Y6Q6Q6Y6的ClpYQ複合體。ClpY 扮演辨識基質並藉著消耗能量將基質解開送入ClpQ 的角色，而ClpQ的功能則是分解基質。關於ClpY如何辨識基質以及如何與之作用機制尚未完全明瞭。 本研究接續前人研究，以大腸桿菌表現蛋白質，以西方墨點法測試ClpY突變株分解基質SulA的能力，MMS (methyl methanesulfonate) test 了解大片段缺失突變對基質辨識與分解基質之能力。結果發現ClpY突變株多無法分解基質，但綜合前人研究顯示只有I domain才是最重要的基質辨識區域。I domain 上的Loop 2 (175-209)為專一辨識與結合的位置並協助基質進入孔洞。此外I domain 突變株， I186N、M187I、A188S、E193L/E194L、Q198L/Q200L、L199Q及N205K 對於基質辨識作用有明顯強弱區別，說明loop 2 對基質辨識的重要性。

ATP-dependent protease generally exist in Bacteria, Archaea and Eucarya. They plays an essential role in controlling the levels of key regulatory proteins and in the elimination of abnormal polypeptides. ClpYQ(HslUV), an (50 KDa) ATPase ClpY and a (19 KDa) peptidase ClpQ, is one of these proteases. ClpYQ is a cylindrical complex, in which two ClpQ hexamers are in an inner core and a hexameric-ClpY is on both side. It is unclear about the mechanisms of how the ClpY recognizes, binds and translocates the specific substrates to ClpQ and the ClpQY degrades the substrates. In this study, using Western blot analysis was used to analyze recognition and binding substrate region in ClpY mutants and MMS test to detect the SulA degradation by ClpY deletion mutants. Most ClpY mutants can not degrade substrate. Compared with previous research, only I domain could be the recognition region. Loop 2 (175-209 aa) in I domain is the specific recognition and binding site also helped delivering substrate into pore site. I domain mutants: I186N, M187I, A188S, E193L/E194L, Q198L/Q200L, L199Q and N205K presented distinguished binding affinity with substrate explained how important loop 2 was.