Analysis of common molecular basis of the AAA ATPase

AAA ATP酶的共同分子基础分析

基本信息

批准号：
18370071
负责人：
OGURA Teru
金额：
$ 11.09万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18370071/
关键词：
AAA ATPase chaperone FtsH p97 fidgetin spastin katanin microtubule ATP依存性プロテアーゼ Cdc48p Katanin

项目摘要

Molecular mechanisms of the AAA ATPase domain of AAA family proteins such as ATP hydrolysis and handling of substrate proteins have been studied. Mutagenesis of residues around the pore region of the hexametric ATPase ring of an AAA protease, FtsH, indicated that some acidic residues are functionally important. We have found that degradation of unfolded polypeptides by some chimeras of FtsH and its C. elegans homologs did not require ATP hydrolysis, and that it was also independent of the conserved aromatic residues located at the pore, suggesting the possibility of energy-independent substrate translocation. We have obtained firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases using mutants of the C. elegans fidgetin homolog, FIGL-1. We have observed that CDC-48.1 and CDC-48.2, p97 homologs in C. elegans, suppress aggregate formation of huntingtin fragments in an ATP-independent manner. Effects of SPAS-1, the C. elegans homolog of spastin, and human katanin on microtubule dynamics have been studied. Overexpression of wild-type SPAS-1 caused disassembly of microtubule network, whereas that of a Walker or pore mutant did not. On the other hand, severing of fluorescently labeled microtubules by human katanin was observed under a fluorescent microscope. It was found that pore mutants of katanin lost the microtubule-severing activity. Processes of microtubule-severing, which was dependent on both katanin and ATP, were observed under a high-speed atomic force microscope.

AAA 家族蛋白的 AAA ATP 酶结构域的分子机制（例如 ATP 水解和底物蛋白处理）已被研究。 AAA 蛋白酶 FtsH 六角 ATP 酶环孔区域周围残基的诱变表明，一些酸性残基具有重要的功能。我们发现，FtsH及其秀丽隐杆线虫同源物的一些嵌合体对未折叠多肽的降解不需要ATP水解，并且它也不依赖于位于孔处的保守芳香族残基，这表明能量无关的底物易位的可能性。我们使用线虫烦躁素同源物FigL-1的突变体，获得了AAA ATP酶亚基间催化ATP水解机制的确凿证据。我们观察到，线虫中的 CDC-48.1 和 CDC-48.2（p97 同源物）以不依赖于 ATP 的方式抑制亨廷顿片段的聚集形成。已经研究了SPAS-1、spastin 的线虫同源物和人katanin 对微管动力学的影响。野生型SPAS-1的过度表达导致微管网络解体，而Walker或孔突变体则不会。另一方面，在荧光显微镜下观察到人剑素对荧光标记的微管的切断。研究发现剑蛋白的孔突变体失去了微管切断活性。在高速原子力显微镜下观察了依赖于剑蛋白和 ATP 的微管切断过程。