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.

我们已经研究了E.c。Elagans中的E. coil和几种AAA蛋白的AAA蛋白酶，并获得了以下结果。FTSH蛋白酶1.确定了FTSH ATPase结构域的晶体结构。 ATPase结构域的六聚体模型支持用于ATP水解的亚基间催化模型。2.FTSH在六聚体的中央孔隙区域中包含高度保守的芳族和甘氨酸残基。我们已经表明，这些残基在蛋白水解及其与ATP水解的耦合中具有重要作用。3。位于中央通道中的酸性残基影响蛋白水解和ATPase活性。4。我们已经建立了一个荧光极化测定系统，以监测底物降解。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。C。Elegrans1.RNAi对paraplegin同源物的RNAi分析显示，表型混合了表型（胚胎致死，幼虫致死和缓慢的生长和缓慢的生长）。10Y47G6A.10，但对Y388f2ar.para没有明显的作用。对于Y47G6A.10，还观察到逐渐延迟的运动性。组织化学和EM分析表明线粒体缺陷2.c。秀丽隐杆线虫具有两个P97/VCP同源物，我们已经表明这些同源物具有必不可少但冗余的功能。3.我们表达了与人体壁肌细胞中GFP融合的PolyQ膨胀。当重复序列长于40时，会形成离散的细胞质聚集体。骨料的形成部分通过两种p97同源物的共表达来部分抑制。4。fidgetin的同源物对于秀丽隐杆线虫中的性腺发生是必不可少的。重组坐立蛋白蛋白被纯化并分析以进行ATPase活性。突变蛋白的体外分析进一步支持AAA蛋白ATP水解的亚基间催化机制。