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Structural and functional studies on AAA proteins in E. coli and C. elegans.

大肠杆菌和秀丽隐杆线虫中 AAA 蛋白的结构和功能研究。

基本信息

批准号：
13480232
负责人：
OGURA Teru
金额：
$ 9.6万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

We have studied on the AAA protease, FtsH, in E. coil and several AAA proteins in C. elagans, and have obtained the following results.FtsH protease1.The crystal structure of the ATPase domain of FtsH was determined. A hexameric model of the ATPase domain supports an inter-subunit catalysis model for ATP hydrolysis.2.FtsH contains highly conserved aromatic and glycine residues in the central pore region of the hexamer. We have shown that these residues have important roles in proteolysis and its coupling to ATP hydrolysis.3.Mutations in acidic residues located in the central channel affected proteolysis and ATPase activity.4.We have established a fluorescence polarization assay system to monitor substrate degradation spectrometrically. Using the system, we have determined the direction of substrate degradation and the energy cost of proteolysis.AAA proteins in C. elegans1.RNAi assays for paraplegin homologs revealed mixed phenotype (embryonic lethal, larval lethal and slow growth) for Y47G6A.10, but no obvious effect for Y38F2AR.para. Progressively retarded motility was also observed for Y47G6A.10. Histochemical and EM analyses indicated mitochondrial defects.2.C. elegans has two p97/VCP homologs, We have shown that these homologs have essential but redundant functions.3.We have expressed polyQ expansions fused to GFP in the body wall muscle cells. When the repeats were longer than 40, discrete cytoplasmic aggregates were formed. The formation of aggregates was partially suppressed by co-expression of either p97 homolog.4.A homolog of fidgetin is essential for gonadogenesis in C. elegans. Recombinant fidgetin proteins were purified and assayed for ATPase activity. In vitro analysis of mutant proteins further supported the inter-subunit catalysis mechanism for ATP hydrolysis by AAA proteins.

本文对大肠杆菌中的AAA蛋白酶FtsH进行了研究。coil和几种AAA蛋白。FtsH蛋白酶1.测定了FtsH的ATP酶结构域的晶体结构。ATP酶结构域的六聚体模型支持ATP水解的亚基间催化模型。2. FtsH在六聚体的中心孔区域含有高度保守的芳香族和甘氨酸残基。我们发现这些残基在蛋白水解及其与ATP水解偶联中具有重要作用。3.位于中央通道的酸性残基的突变影响蛋白水解和ATP酶活性。利用该系统，我们确定了底物降解的方向和蛋白质水解的能量消耗。paraplegin同源物的RNAi分析揭示了Y47G6A.10的混合表型（胚胎致死、幼虫致死和缓慢生长），但对Y38F2AR没有明显影响。组织化学和电镜分析显示线粒体缺陷。3.我们在线虫体壁肌细胞中表达了与GFP融合的polyQ扩增产物。当重复序列超过40个时，形成离散的细胞质聚集体。p97同源物的共表达可部分抑制聚集体的形成。优美的纯化重组fidgetin蛋白并测定ATP酶活性。对突变蛋白的体外分析进一步支持了AAA蛋白ATP水解的亚基间催化机制。