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.

我们对大肠杆菌中的AAA蛋白酶FtsH和线虫中的几种AAA蛋白进行了研究，得到了以下结果：FtsH蛋白酶1.确定了FtsH的ATPase结构域的晶体结构。 ATPase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。我们已经证明这些残基在蛋白水解及其与 ATP 水解的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。4.我们建立了荧光偏振测定系统，以光谱法监测底物降解。使用该系统，我们确定了底物降解的方向和蛋白水解的能量成本。秀丽隐杆线虫中的 AAA 蛋白。paraplegin 同源物的 RNAi 测定揭示了 Y47G6A.10 的混合表型（胚胎致死、幼虫致死和生长缓慢），但是对Y38F2AR.para无明显影响。还观察到 Y47G6A.10 的运动逐渐迟缓。组织化学和电镜分析表明线粒体缺陷。2.C．线虫有两个p97/VCP同源物，我们已经证明这些同源物具有必要但多余的功能。3.我们在体壁肌肉细胞中表达了与GFP融合的polyQ扩展。当重复次数超过 40 时，就会形成离散的细胞质聚集体。 p97 同源物的共表达部分抑制了聚集体的形成。4. fidgetin 同源物对于秀丽隐杆线虫的性腺发生至关重要。纯化重组坐立不安蛋白并测定 ATP 酶活性。突变蛋白的体外分析进一步支持了 AAA 蛋白水解 ATP 的亚基间催化机制。