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

本研究对大肠杆菌中的AAA蛋白FtsH和角叉菜中的几种AAA蛋白进行了研究，得到了以下结果：1.确定了FtsH的ATPase结构域。ATPase结构域的六聚体模型支持ATP水解亚单位间催化模型。2.FtsH在六角体的中心孔区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质降解及其与ATP水解酶的偶联过程中起着重要的作用。3.位于中心通道的酸性残基的突变影响了蛋白质的降解和ATPase的活性。4.我们建立了一套荧光偏振分析系统来检测底物的降解情况。利用该体系，我们确定了底物降解的方向和蛋白降解的能量成本。对雅典娜1号截瘫蛋白同源物的RNAi分析显示，Y47G6A.10表现为混合表型(胚胎致死、幼虫致死和缓慢生长)，而Y38F2AR没有明显的影响。Y47G6A.10也观察到进行性运动迟缓。组织化学和EM分析表明线虫存在线粒体缺陷。2.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融合GFP的多聚Q扩增产物。当重复数大于40时，形成离散的细胞质聚集体。聚集体的形成被p97同源物的共表达部分抑制。4.fidgetin的同源物对于线虫的性腺发生是必不可少的。纯化重组fidgetin蛋白，并测定其ATPase活性。对突变蛋白的体外分析进一步支持了AAA蛋白对ATP水解的亚基间催化机制。

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.