大腸桿菌ClpYQ蛋白酶可能基質之研究

Abstract

蛋白質的降解為調控生物體中蛋白體組成的重要機制，而ATP依賴型蛋白酶即是負責蛋白質降解的重要一員。在大腸桿菌中，ClpYQ蛋白酶為三個主要的ATP依賴型蛋白酶之一，其由具有ATPase活性的ClpY和具有分解酶活性的ClpQ所組成。除了分解菌體內摺疊錯誤的蛋白質之外，ClpYQ還會在必要情況下分解具有活性的調控蛋白以改變細胞的生理狀態，使其得以適應外在環境條件。已知的ClpYQ的基質包含SulA、RcsA、RpoH、TraJ及RNase R。 由於ClpY在ClpYQ的作用過程中負責辨認、解開，和轉送基質進入負責分解的ClpQ活性位，因此能與ClpY產生交互作用的蛋白即有可能為ClpYQ的基質。由大腸桿菌K12蛋白質晶片分析結果發現大腸桿菌體內六個功能各異的蛋白質YbaB、YfbV、RbsD、CobB、RhlB和YgbT，與ATPase ClpY之間有較強的交互作用。本研究試圖確認此六個蛋白質是否為ClpYQ的基質，首先以酵母菌雙雜交系統和胞內共沉澱試驗測定ClpY和此六個蛋白之間的交互作用，並針對其中作用力最強之YbaB蛋白進行Kd值測定。基質確認部分則分別以胞外和胞內降解試驗分析ClpYQ是否會分解這六個蛋白質。 酵母菌雙雜交系統及胞內共沉澱試驗結果共確定YbaB、RhlB和YfbV與ClpY具有交互作用。以胞外和胞內的降解試驗所進行之基質確認結果發現MBP-YbaB、MBP-YgbT和MBP-RbsD可在胞外降解試驗中被ClpYQ分解，而MBP-CobB和MBP-RhlB則可在胞內降解試驗中觀察到分解情形。此外，MBP-YfbV在胞內共沉澱試驗中被確認為ClpXP之基質，然而其被ClpXP分解之過程會受ClpYQ影響。

Proteolysis is a vital mechanism to regulate the cellular proteome in all kingdoms of life, and ATP-dependent proteases take a crucial role within that process. In Escherichia coli, ClpYQ is one of the three primary ATP-dependent proteases. ClpYQ contains ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), the peptidase responsible for proteolysis. Besides removals of abnormal peptides in cell, ClpYQ degrades regulatory proteins if necessary and thus let cells adjust to various environmental conditions. In E. coli, SulA, RcsA, RpoH and TraJ as well as RNase R, have been identified as natural protein substrates of ClpYQ. As the ATPase ClpY is responsible for protein recognition, unfolding, and translocation into the catalytic core of ClpQ, the interactions with ClpY suggested previously unknown substrates of ClpYQ. According to the analysis of protein interaction using E. coli K12 proteome chips, there are six proteins, YbaB, YfbV, RbsD, CobB, RhlB, and YgbT, interacting strongly with ClpY. With an attempt to find novel substrates of ClpYQ, various methods were used to verify the six putative substrates in this study. The yeast two-hybrid assay and in vivo pull-down assay were performed to confirm the interactions with ClpY, and the YbaB protein, which has the strongest interaction, was chosen to determine the Kd value. In vitro degradation and in vivo degradation were carried out for the direct observations of degradation. The results of yeast two-hybrid assay and pull-down assay showed that YbaB, RhlB, and YfbV do have interactions with ClpY under different conditions. In the test of degradation, MBP-RbsD and MBP-YbaB and MBP-YgbT could be degraded by ClpYQ in vitro while MBP-CobB and MBP-RhlB have shown decay in vivo. In addition, the results of in vivo degradation indicated that MBP-YfbV is the substrates of ClpXP, but the proteolysis process is likely to be affected by ClpYQ.