大腸桿菌ClpQ蛋白分子之間的聚合和其C端的功能

ClpQ subunit oligomerization and its C domain function in Escherichia coli

林佩宜(Pei-I Lin)

指導教授 ： 吳蕙芬

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01529

摘要

大腸桿菌ClpQY (HslVU)是一個ATP依賴的蛋白酶，ClpQY蛋白酶包含了具ATPase活性的大蛋白分子和一個小分子的peptidase，ClpQY蛋白酶作為一伴隨蛋白質 (Chaperon) 扮演分解錯誤摺疊的蛋白質，也為一催化分解不正常的蛋白質的蛋白酶角色。ClpQ和ClpY分子由六個單元體，形成一六元環狀結構，ClpQ和ClpY分別由兩個六元環形成一像啞鈴形狀的蛋白質複合體，單一ClpY六元環結合在兩個六元環堆疊成ClpQ的兩側。本篇研究重點在ClpQ單元體間的作用如何形成穩定六元環結構 (oligomerization) 和探討ClpQ C端對ClpQY蛋白酶活性的影響。利用綠膿桿菌的clpQ基因部分區段與大腸桿菌的clpQ基因部分區段做融合的建構，以進行作用區段的確認，實驗得知ClpQ C端後段在其單元體間的作用與功能上的重要性比其中的一段二级螺旋結構 (O-helix) 顯得重要；另外再對C端區段進行隨機點突變，並以ClpQY的基質RcsA對cpsB10-lacZ正向調控的系統進行β-galactosidase的活性測試，和以MMS誘導ClpQY基質SulA觀察對細胞生長的影響結果，藉此分析ClpQ的C端是否負責在大腸桿菌中對其本身具有專一性的辨認能力；使用yeast two-hybrid系統偵測ClpQ突變株之間交互作用的情形幫助了解單元體oligomerization的穩定性。結果顯示ClpQ C端上的突變株對ClpQY蛋白酶功能上的影響並未能與其單元體之間的作用能力有一定的正相關。靠近ClpQ C端後段的三個突變位置Asp154 (D154N)，Cys160 (C160A) 和Tyr162 (Y162A)在與ClpQ/ClpQ聚合一穩定結構的環境下使得ClpQY 蛋白酶活性功能受損。

關鍵字

蛋白酶大腸桿菌 ； 綠膿桿菌 ； 伴隨蛋白 ； 基質

並列摘要

Abstract

The E. coli ClpQY (HslVU) is an ATP-dependent protease that consists of an ATPase large subunit and a peptidase small subunit. The function of ClpQY protease is as a chaperon in refolding of misfolded peptides and as a protease to catalyse the degradation of abnormal proteins as well. Six identical subunits of both ClpQ and ClpY self-assemble into an oligomeric ring and two rings of each subunit form a dumbbell shape complex, two ClpQ rings surrounded by two ClpY single rings. Construction of the fusion gene of P. aeruginosa and E. coli clpQ was used to analyse the C domain and O-helix within C domain of ClpQ for subunits oligomerization and ClpQY protease function. The further C-terminal region of ClpQ is more important in proteolytic function than O-helix which is likely important to ClpQ subunits oligomerization. The C domain region of ClpQ was randomly mutagenized and the single point mutants were selected for investigation of their in vivo function. In addition, The β-galactosidase assay of which the reporter gene (cpsB10 – lacZ) was activated by RcsA, the substrate of ClpQY, and the accumulation of cell division inhibitor, SulA, which increased the sensitivity to MMS induction of E. coli cells were both used to detect ClpQY protease function. Using yeast two-hybrid system, I explore the in vivo protein-protein interactions of the individual mutant subunits of ClpQ involved in self-oligomerization. Several mutants altering ClpQY protease activity were verified. Mutants Asp154 (D154N), Cys160 (C160A) and Tyr162 (Y162A) carrying mutation located in the C– terminal region of ClpQ and have defective proteolytic function based on cpsB10-lacZ expression and MMS test.

並列關鍵字

ClpQY ； HslVU ； protease ； chaperon ； E. coli ； P. areuginosa ； ClpQ ； RcsA ； SulA

參考文獻

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