大腸桿菌ClpYQ辨識基質SulA羧基端之研究
Other Title
The ＂Escherichia coli＂ ClpYQ Protease Recognizes the C-terminal Tail of SulA
Journal
臺灣農業化學與食品科學
Journal Volume
52
Journal Volume
52
Journal Issue
1
Journal Issue
1
Pages
48-58
Start Page
48
End Page
58
Date Issued
2014-02
Author(s)
Abstract
大腸桿菌ATP依賴型蛋白酶在細菌體進行自身蛋白質品質調控過程中扮演重要的角色，以ATP的水解做為能量的來源，將具有危害性的蛋白質分解，來維持細胞本身的正常生理功能。ClpYQ蛋白酶為ATP依賴型蛋白酶之一，透過ClpY進行基質的辨識、結合、解構及轉送入ClpQ蛋白酶中被分解，SulA為具有抑制細胞分裂功能之蛋白質，於細菌SOS反應下會被大量誘導表現，以避免受損的DNA傳遞到子代。當DNA修復完成後，SulA可被細胞質內的蛋白酶分解，讓細菌重新恢復進行細胞分裂，對SulA進行分解之蛋白酶有Lon和ClpYQ蛋白酶。於細胞內觀察ClpY與C端缺失之SulA突變蛋白交互作用，發現SulA 141-150胺基酸區域對ClpY的辨識具有專一性，於革蘭氏陰性菌中，SulA位於C端142至147胺基酸，G^(142)F^(143)I(144)M^(145)R^(146)P^(147)，具有序列保守性，因此，將當中疏水性胺基酸I^(144)進行單一定點突變，將其由非極性異白胺酸(Ile)置換為帶極性天門冬醯胺(Asn)，SulA*I144N點突變蛋白質與MBP（maltose-binding-protein）融合，於細胞內或細胞外偵測此一融合蛋白質被ClpYQ蛋白酶分解之情形。試驗結果顯示，MBP-SulA*I144N，具較長半衰期，較不易被ClpYQ蛋白酶分解，由於MBP-SulA*I144N不具有抑制細胞分裂之活性，顯示I144N會改變SulA之生理功能。因此，SulA第141-150個胺基酸片段內具保守性之疏水性胺基酸，對於被ClpY辨識及結合具有重要性，且僅改變其中單胺基酸即會影響SulA的生理活性。
The ＂Escherichia coli＂ ATP dependent proteases play several important roles in protein quality control and they use the energy from ATP hydrolysis to recognize and degrade the abnormal proteins. ClpYQ protease, one of the ATP dependent proteases, is a two component complex. ClpY recognizes, unfolds and translocates the natural substrates into the catalytic core site of ClpQ for the degradation. The cell division inhibitor, SulA, induced during an SOS response, prevented a cell division. However, both Lon and ClpYQ proteases were capable of degrading it and that could restore the normal cell growth. In this study, through the in vitro assays, the 141 to 150 residues of SulA were shown necessary for interaction with ClpY. There is a conserved region between 142 to 147 residues, G^(142)F^(143)I^(144)M^(145)R^(146)P^(147), in SulA, from Gram-negative bacteria. SulA*I144N point mutant, with a substitution of Asn for Ile, was further used for detection of its degradation with ClpYQ protease. MBP(maltose-binding-protein)-SulA*I144N has a longer half-life than that of MBP-SulA in the presence of ClpQ and ClpY. Moreover, SulA*I144N mutant has no inhibitory activity on cell division; this mutant has an effect on the function of SulA. Our results suggested that the conserved hydrophobic region in SulA is necessary for its inhibitory activity as well as for an association with ClpY for its degradation by ClpYQ protease.
Subjects
ATP依賴蛋白酶
基質辨識
翻摺
轉送
分解
ATP-dependent protease
substrates recognition
unfolding
translocation
degradation
Publisher
臺灣農業化學會
臺灣食品科學技術學會
Type
journal article