大腸桿菌莢膜生合成調節蛋白RcsA為ClpYQ蛋白酶的基質之研究

ATP 依賴型蛋白酶可利用水解 ATP 所產生的能量，分解細胞中摺疊錯誤或不需要的蛋白質，維持細胞正常的生長與代謝。大腸桿菌之 ClpYQ 蛋白酶是由 ClpY 與 ClpQ 所組成雙元體蛋白酶 (two-component protease)，ClpY 具有ATPase 的活性，可辨認、拆解目標基質，並將此多肽鏈傳遞至 ClpQ 的活性部位中進行分解。RcsA 與 RcsB 蛋白為莢膜多醣生合成基因 (cps gene) 之調控蛋白，可促使胞外多醣的產生，研究指出胞外多醣與細菌在宿主外之存活能力、抵抗外在乾燥環境的耐受性，以及大腸桿菌生物膜的形成有關。於前人研究中，以lon- 突變株建構cpsB::lacZ 報導基因，發現 ClpYQ 蛋白酶可藉由調節細胞中RcsA 的量，影響cpsB::lacZ 的表現量。在本篇實驗中以 MBP (maltose binding protein) 與 RcsA 建構融合蛋白，希望藉由此融合蛋白反應細胞中 RcsA 與 ClpYQ 蛋白酶之間的交互作用。結果顯示，在低溫下大量表現 ClpYQ 蛋白酶可使 MBP-RcsA 的累積量減少，在半衰期試驗中，偵測到高溫下在lon- clpQ+Y+ 菌株中之 MBP-RcsA 半衰期約30分鐘，接著於in vivo 及in vitro 之親和力試驗中，觀察到 ClpY 可和 MBP-RcsA結合；並於in vitro 下，添加 ATP，發現 MBP-RcsA 可被 ClpYQ 蛋白酶分解。這些結果顯示，MBP-RcsA 為大腸桿菌 ClpYQ 蛋白酶之基質。但當進一步探討在胞內之 RcsB 蛋白是否會影響蛋白酶分解 MBP-RcsA 時，發現有其他的蛋白酶在低溫下可以調節 MBP-RcsA 在細胞中的累積量。

關鍵字

RcsA ； ATP 依賴型蛋白酶ClpYQ 蛋白酶RcsB ； ycbZ

並列摘要

Proteases play an important role in metabolic regulation. They degrade many abnormal proteins and regulatory proteins and thus let cells adjust to various environmental conditions. In Escherichia coli, ClpY or ClpQ oligomerizes as a hexamer,and four hexamers organize a complex. ClpY recognizes, unfolds the substrate, and then transfers the polypeptide into the catalytic core of ClpQ. ClpQ degrades the substrates and releases the remaining peptides. RcsA and RcsB are positive activators for transcription of cps (capsular polysaccahride synthesis) genes. This exopolysaccahride is essential for resistant to desiccation, the later stages of E. coli K-12 biofilm development and the lifestyle outside the host. In a previous study, overproduction of ClpYQ represses cpsB::lacZ expression, but there has been no direct observation demonstrating that ClpYQ degrades RcsA. In this study, we constructed MBP-RcsA fusion protein to imitate unstable chromosomal RcsA protein. Pull-down and half-life assay wrer used to observe the relationship between MBP-RcsA and ClpYQ protease. The measurement of MBP-RcsA half-life was performed in lon- and lon- clpY- clpQstrain. Compared to lon- mutant, turnover rate of MBP-RcsA in lon- clpY- clpQ- strain was relatively slow and MBP-RcsA was stable. In vivo and in vitro pull-down assays demonstrated that ClpY was pulled down with MBP-RcsA. In vitro, the more rapid degradation of MBP-RcsA was observed in the presence of ClpYQ protease at 41°C. In the absence of RcsB at 30°C, the half-life of MBP-RcsA became shorter. These results indicate that MBP-RcsA is substrate of ClpYQ protease, also there is another protease which could degrade MBP-RcsA in the absence of RcsB.