大腸桿菌熱休克蛋白酶ClpYQ之基質SulA被辨識區域特性之研究

Abstract

大腸桿菌中ClpYQ蛋白酶為一種ATP依賴蛋白酶，由具有ATPase及unfoldase活性的ClpY，與具有peptidase活性的ClpQ所構成的雙單元體。這類蛋白酶在細胞中，可降解構形錯誤或是具危害性的蛋白質，以維持細胞正常生理作用，避免細胞受到危害。在ClpYQ蛋白酶中，ClpY會負責辨識基質，並水解ATP作為能量來源，將基質結構打開並傳送至ClpQ的活性區，以進行降解作用。然而，關於ClpYQ蛋白酶是如何選擇辨識基質，及後續降解作用的詳細機制，目前仍不清楚。SulA是一個細胞分裂的抑制物，當細胞暴露在逆境下時會產生SOS反應，誘導大量SulA蛋白表現，以避免受損的DNA傳到子代細胞。目前已知可分解SulA的蛋白酶為Lon及ClpYQ，其中又以Lon為主要負責分解的蛋白酶。之前有研究指出，Lon可以藉由辨認SulA之C-端末8個胺基酸，與之結合並將其降解，但是ClpYQ卻不能。對於ATP依賴蛋白酶來說，為避免不必要的降解，如何選擇辨認需要降解的基質是非常重要的。本實驗中，為確認ClpYQ蛋白酶辨認SulA蛋白的區域，建構SulA之C-端不同大小片段缺失的突變蛋白，以酵母菌雙雜交系統測試，各個SulA突變蛋白與ClpY之間交互作用的情形，發現ClpY辨識的區域可能位於C-端的高疏水性片段，C-端第20 - 30個胺基酸。於此區域內再建構點突變蛋白，分析不同性質的胺基酸對於ClpY交互作用的影響，結果顯示當點突變取代為親水性胺基酸時，會降低SulA蛋白與ClpY之間的交互作用。之後測試各個SulA突變蛋白的活性表現，及被ClpYQ蛋白酶降解之情形，結果顯示SulA之C-端第20 - 45個胺基酸的區域，對於其抑制細胞分裂的活性表現是重要的，且對於ClpYQ蛋白酶的降解作用也會造成影響，因此ClpYQ蛋白酶應可藉由SulA蛋白之活性表現與否，來辨別其是否需要降解。

ClpYQ is an ATP-dependent protease from Escherichia coli and a two component complex composed of ClpY, which is an ATPase and unfoldase, and ClpQ peptidase. Degradation of denatured or damaged proteins by this proteases helps protect the normal cell growth from the harmful effects of these proteins. The ClpY is thought to recognize protein substrates, denature them, and translocate the unfolded polypeptide into the catalytic cavity of the ClpQ for degradation. However, little information is available on the recognition of substrates for ClpYQ and on the mechanism by which they were selected, unfolded, and translocated by ClpY to the interior of the ClpQ. SulA, induced in the SOS response, is a cell division inhibitor and prevents the distribution of damaged DNA into daughter cells during DNA repair processes. SulA can be degraded by ATP-dependent proteases such as Lon and ClpYQ, and the degradation in vivo seems to be predominantly by Lon, while ClpYQ appears to act as a backup for Lon. It was reported previously that the region of C-terminal eight amino acid residues of SulA was essential for interaction with Lon but not with ClpYQ. To avoid unnecessary degradation of cellular proteins, substrate selection by ATP-dependent proteases is tightly regulated; therefore, it is interesting to investigate the recognition region of SulA by ClpYQ. In this study, the deletion mutants of SulA with regard to C-terminus were constructed and the interaction with ClpY was analyzed in yeast two-hybrid system. The results showed that ClpY recognized the hydrophobic region of SulA, C20 - 30 aa, and the recognition was also likely to rely on hydrophobic interaction following the observation that the binding activity decreased if the substituted residue was polar. The C-terminal region of SulA, C20 - 45 aa, seemed to be important for its activity with an inhibition of cell division, and the region is necessary for the degradation by ClpYQ. Therefore, ClpYQ protease would be able to distinguish whether SulA is to be degraded by the activity of inhibition.