Please use this identifier to cite or link to this item:
標題: 第二型蛋白分泌機器ATPase分子機制之探討(I)
Molecular Characterizations of the Type II Protein Secretion Atpase (I)
作者: 胡念台
關鍵字: 生物技術
摘要: 格蘭氏陰性菌利用第二型分泌機器 (T2SS) 將胞外蛋白自胞質週緣區運送通過外胞膜,經此分泌機器送至胞外的蛋白質被作為攻擊動物或植物細胞之用,或為細菌在不同自然環境生存提供養分。T2SS由 12-15個蛋白質組成,其中一個外胞膜蛋白GspD (secretin) 為構成分泌孔道的主要成分;超低溫電子顯微鏡結構分析顯示GspD複合體呈現12位元對稱,具有一個兩端對外開放中央被擋住的空腔,彷彿一個被封住的孔道。推測此類似封閉的孔道可能經由一種類似第四型纖毛 (T4p) 的結構 (pseudopilus) 在施力情況下將其推開,位於孔道一端開口處的被分泌蛋白則同時被推出胞外。由纖毛蛋白 (pilin) 組裝成的T4p是位於細菌表面細長形的絲狀構造,兼具強度與彈性,為細菌附著於宿主細胞表面或在表面移動時所必須。T2SS需要 4-5 個類似纖毛蛋白的成分,稱作類纖毛蛋白 (pseudopilins),其中之一 GspG為pseudopilus的主要組成蛋白。T4p組裝機器與T2SS具有幾個相似的成分,除了外胞膜蛋白 GspD/PilQ,還包含位於胞內的 ATPase GspE/PilB,以及一個穿膜多次的內胞膜蛋白 GspF/PilC,ATPase 可能為能量提供者,穿膜多次的內胞膜蛋白的功能則尚不清楚。此外,T2SS還需要 3 個內胞膜蛋白 GspL, -M, -C,十字花科黑腐病菌 T2SS中的 GspL, -M, -C 形成複合體,作為胞內 ATPase 以及位於外胞膜的分泌孔道間的橋樑,推測可能扮演能量傳輸者的角色;免疫共沉澱實驗結果顯示GspD 與 GspC 有相互作用關係,而文獻中已有大量數據顯示 GspE ATPase 透過與 GspL N端 (GspLN) 朝向胞內區域的結合附著於內胞膜。本實驗室最近發現 GspE 必須與 ATP 結合方可與 GspLN 結合,但不需要GspE 水解 ATP;然而,失去水解 ATP 能力的突變 GspE 蛋白卻不具分泌功能,說明需要GspE 水解 ATP 的步驟可能位於 GspE 與 GspLN 結合之後。GspE 本身的 ATPase 活性非常低,近日文獻指出 GspE ATPase 活性受 GspLN 與磷脂 (phospholipids) 的共同刺激而顯著提升,暗示 GspE ATPase 的活化可能受到嚴密而複雜的調控。為增進對GspE 在T2SS中功能的了解,本計畫打算針對十字花科黑腐病菌的 XpsE 蛋白進行詳細的生化、結構以及遺傳分析,以期達成三項目標: 1) 了解細胞膜在調控 XpsE 蛋白於T2SS中行使功能時可能扮演的角色; 2) 藉由野生型蛋白構形的分析,及與已知詳細生化特性的突變 XpsE 蛋白構形的比較,深入了解 XpsE在T2SS中的運作機制; 3) 了解 XpsE如何將水解 ATP 產生的化學能轉化為促使被分泌蛋白運送至胞外的機械能。
Type II secretion system (T2SS) is utilized by a wide variety of Gram-negative bacteria for transporting across outer membrane proteins that are required for attacking animal or plant cells or for survival in different environmental niches. The secretion machinery comprises of 12-15 protein components. An outer membrane protein GspD (secretin) is the basic component of the secretion pore. As revealed by cryoEM, the GspD complex exhibits a structure of 12-fold symmetry with a central cavity open at two ends and blocked in the middle. A type IV pilus-like (pseudopilus) structure was postulated to exert pushing force driving secreted protein through the closed pore by extending from cytoplasmic membrane. The type IV pilus (T4p) comprising of pilin is the long, thin filament appearing on bacterial cell surface that exhibits strength and flexibility for adhesion and twitching motility. Of four or five pilin-like proteins (pseudopilins) that are essential for T2SS, GspG is known to be the major constituent of pseudopilus. The machinery for assembling type IV pilus (T4p) has several components in common with T2SS. In addition to secretin GspD/PilQ, a cytoplasmic ATPase GspE/PilB and a polytopic cytoplasmic membrane protein GspF/PilC are essential for T2SS/T4p assembly. While the ATPase may act as energy supplier for protein secretion or assembly of pseudopilus/pilus, functional role of the polytopic cytoplasmic membrane protein remains elusive. Moreover, three additional cytoplasmic membrane proteins GspL, -M, -C are essential in T2SS. A GspL-M-C ternary complex demonstrated in the Xanthomonas campestris T2SS was postulated to act as energy transducer, supported by its bridging between the cytoplasmic ATPase and the secretion pore in outer membrane. Interaction between secretin GspD and GspC was suggested with coimmune precipitation data, whereas GspE ATPase is well known to be associated with membrane by its interaction with the N-terminal cytoplasmic domain of GspL (GspLN). Recently, we demonstrated that association of GspE with GspLN is made possible by ATP binding to GspE, not requiring ATP hydrolysis. However, mutant GspE deficient in ATPase activity is not functional in protein secretion, suggesting ATP hydrolysis may be essential for steps downstream of GspE-GspL association. GspE exhibits very weak basal ATPase activity. Nevertheless, it was lately shown to be stimulated to much higher level by GspLN and phospholipids, implying precise and elaborate regulatory mechanism may be involved. To improve our understanding of the functional role of GspE in T2SS, we propose to characterize the X. campestris XpsE by taking biochemical, structural and genetic approaches with three specific aims: 1) To comprehend the roles that membrane may play in modulating XpsE function in T2SS; 2) To gain information on how XpsE operates mechanically in T2SS from its structural alteration that may be caused by mutation of conserved residues; 3) To comprehend how chemical energy generated from ATP hydrolysis by XpsE is transformed into mechanical force driving secreted protein through secretion pore.
其他識別: NSC97-2321-B005-011
Appears in Collections:生物化學研究所



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.