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標題: E395Q突變對XpsE蛋白聚合的影響
Influence of E395Q mutation on XpsE protein oligomerization
作者: 李鳴亞
Lee, Ming-Ya
關鍵字: XpsE protein
出版社: 生物化學研究所
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(1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol, 174, 2679-2687. Hu NT, Leu WM, Lee MS, Chen A, Chen SC, Song, YL and Chen, LY (2002) XpsG, the major pseudopilin in Xanthomonas campestris, pv. campestris, froms a pilus-like structure between cytoplasmic and outer membranes. Biochem J, 365, 205-211. Kao KM (2005) Type II secretion apparatus of Xanthomonas campestris: Determination of ATPase activity of XpsE and mutant analysis of XpsE with mutations at nucleotide-binding motifs. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing, Taichung, Taiwan, R. O. C. Lee MS, Chen LY, Leu WM, Shiau RJ and Hu NT. (2005) Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis J Biol Chem, 280, 4585-91. 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Walker JE, Saraste M, Runswick MJ and Gay NJ (1982) Distantly related sequences in the alpha- and beta-subunit of ATP synthase, myosin, kinased and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J, 1, 945-951 Wallden K, Calzada AR and Waksman G. (2010) Type IV secretion systems: versatility and diversity in function. Cell Microbiol, 12, 1203-1212. Yamagata A and Tainer JA (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J, 26, 878-890. Zhu ZL (2006) Type II Secretion Apparatus of Xanthomonas campestris : Expression and Functional Characterization of the Cytoplasmic Domains of XpsF. Master thesis. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C.
摘要: 格蘭氏陰性菌利用第二型分泌系統 (T2SS) 將蛋白由內外膜的夾層periplasm分泌到胞外。十字花科黑腐病菌的T2SS至少由12個蛋白所組成,其中XpsE蛋白為唯一的胞內蛋白。過去研究顯示XpsE蛋白可以藉著和ATP結合形成多倍體,而XpsE蛋白的多倍體藉著和內膜蛋白XpsL結合完成分泌。在XpsE蛋白及其同源性蛋白中有四個保留性非常高的序列,分別為Walker A box、Asp box、Walker B box及His box,在霍亂弧菌N端缺損90個胺基酸的EpsE蛋白晶體結構中,這四個保留性胺基酸序列均位在ATP周圍,其中最接近ATP的胺基酸,對應到XpsE分別為K331, E357, E395及H420,突變株的分析顯示這四個胺基酸為XpsE行使正常分泌所必需。過去的實驗發現將XpsE蛋白的E395突變成Glutamine (E395Q),XpsE會傾向於維持多倍體的狀態,不易解離成單體。為進一步探討E395在XpsE蛋白形成多倍體或其解散中的重要性,本研究利用大腸桿菌大量表現XpsEE395Q-Strep突變蛋白,將經過親和性管柱純化的XpsEE395Q蛋白,經由分子篩管柱層析 (gel filtration chromatography) 或分析式超高速離心 (analytical ultracentrifugation,AUC) ,分析XpsEE395Q蛋白分子大小,結果發現E395Q的突變不但使XpsEE395Q蛋白不易解離成單體,其分子大小也和正常的XpsE蛋白不相同,AUC 分析結果顯示正常型XpsE呈現單體、三倍體與六倍體,而XpsE (E395Q) 則呈現二倍體及其倍數的多倍體。此外,在十字花科黑腐病菌 (Xanthomonas campestris) 中表現帶有ECFP螢光蛋白的XpsEE395Q-ECFP,在螢光顯微鏡中觀察XpsEE395Q-ECFP的分佈情形,發現表現正常且帶有ECFP的XpsE蛋白,在外膜分泌孔道缺失時,呈現點狀聚集,推測可能代表XpsE在分泌完成前所處的狀態,而觀察XpsEE395Q-ECFP則無點狀聚集訊號,說明XpsEE395Q形成異常的多倍體,且此多倍體在胞內無法聚集至第二型分泌系統其他組成蛋白所在位置。
The type II secretion system (T2SS) is utilized by Gram-negative bacteria for secreting protein from the periplasm to extracellular space. The Xanthomonas campestris T2SS is composed of at least 12 proteins. XpsE protein is the only cytosolic component. Previous research showed that XpsE protein can form oligomer triggered by its binding to ATP. The oligomeric XpsE protein can then interact with the inner membrane protein called XpsL for completing the secretion process. The XpsE protein and its homologous proteins have four highly conserved amino acid sequences. They are Walker A box, Asp box, Walker B box and His box. As revealed in crystal structure of the Vibrio cholerae T2SS ATPase EpsE, whose N terminal 90 amino acids were deleted, these four conserved sequences are surrounding the bound ATP. And the amino acids in these four conserved sequences of XpsE closest to ATP are K331, E357, E395 and H420. Mutant analysis of these four amino acids suggested that they are essential for normal function of XpsE. Previous study has shown when E395 was mutated to Glutamine (E395Q), the mutated XpsE protein tends to stay as non-dissociable oligomer. To understand the significance of E395 in XpsE oligomer formation and dissociation, I purified the XpsEE395Q-Strep protein overexpressed in E.coli by affinity chromatography. Purified XpsEE395Q protein was then analyzed for size distribution by using gel filtration chromatography and analytical ultracentrifugation (AUC). I found the E395Q mutation not only makes XpsE protein prone to be oligomeric but also exhibit oligomeric states differing from those of the wild-type XpsE protein. The result of AUC showed the wild-type XpsE protein appeared as monomer, trimer and hexamer, whereas XpsEE395Q protein as dimer and its multimers. To monitor oligomer formation in vivo, I also expressed XpsEE395Q in fusion to ECFP (a fluorescent protein) from plasmid-encoded gene in X. campestris. Previous studies suggested when expressed in xpsD-null strain, the wild-type XpsE-ECFP forms foci at cell boundary, implicating a pre-secretion state of XpsE protein. Here I found no foci formation by expressing plasmid-encoded XpsEE395Q-ECFP in xpsE-, xpsD- double mutant strain. In summary, the results from this study suggested that E395Q mutation caused XpsE protein form non-dissociable dimer (and its multimers) that can not be assembled to cell boundary where the T2SS machine is located.
其他識別: U0005-2107201111391700
Appears in Collections:生物化學研究所



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