Analysis of common molecular basis of the AAA ATPase

研究了AAA家族蛋白的ATP酶结构域的分子机制，如ATP水解和底物蛋白的处理。AAA蛋白酶FtsH的六维atp酶环孔区周围残基的突变表明，一些酸性残基在功能上很重要。我们发现，FtsH及其线虫同源物的一些嵌合体降解未折叠的多肽不需要ATP水解，而且它也不依赖于位于孔处的保守芳香残基，这表明可能存在能量独立的底物易位。我们已经获得了确凿的证据，证明了3a ATP酶在亚基间催化ATP水解的机制，使用的是秀丽隐杆线虫的fidgetin同源突变体，如图1所示。我们观察到线虫的CDC-48.1和CDC-48.2， p97同源物，以atp不依赖的方式抑制亨廷顿蛋白片段的聚集形成。研究了SPAS-1、线虫的spastin同源物和人类的katanin对微管动力学的影响。野生型SPAS-1的过表达会导致微管网络的解体，而Walker或孔突变体则不会。另一方面，在荧光显微镜下观察到荧光标记的微管被人角朊蛋白切断。结果发现，卡他蛋白的孔突变体失去了切断微管的活性。在高速原子力显微镜下观察了同时依赖于katanin和ATP的微管切断过程。

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.