Molecular, cellular, and developmental studies on AAA chaperones

研究了AAA伴侣蛋白常见活性的分子机制，如底物蛋白的ATP水解性和转位。我们提出了AAA-ATPase对ATP的亚基间催化机制和底物转位的线索模型，并得到了支持这些模型的证据。对AAA酶FtsH的六聚体ATPase环孔内的保守残基进行了突变，结果表明，保守的芳香残基可能起到底物结合和转位的作用。研究发现，FtsH可以通过将一个内部环以依赖于ATP的方式移位到蛋白酶小室来启动黄曲霉毒素的蛋白降解。我们还发现，FtsH及其线虫的一些嵌合体对未折叠多肽的降解不需要ATP水解酶。利用线虫fidgetin同源物的突变体，已经获得了AAA ATPase对ATP亚基间催化机制的确凿证据，图1。我们还研究了一些与人类遗传病致病因子同源的优雅线虫AAA蛋白的细胞功能。研究发现，p97与Paget病和额颞部痴呆相关的包涵体肌病相关，在精子发生向卵子发生的转换、减数分裂I和ERAD的进展中起重要作用。我们观察到，p97在线虫中的过表达在体内部分抑制了多谷氨酰胺聚集体的形成，而纯化的p97在体外以一种不依赖于ATP的方式抑制了Huntingtin片段的聚集体形成。我们已经展示了p97的六个Ubx接头蛋白在线虫中的时空差异表达模式。我们发现，与遗传性痉挛截瘫相关的线虫spastin同源基因spas-1的过度表达导致微管网络的解体，表明spas-1参与了微管动力学。

Molecular mechanisms of common activities of AAA chaperones such as ATP hydrolysis and translocation of substrate proteins have been studied. We have proposed an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases and a threading model for substrate translocation, and obtained supporting evidence for these models. Mutagenesis of conserved residues at the pore of the hexameric ATPase ring of an AAA protease, FtsH, indicated that the conserved aromatic residue is suggested to act for substrate binding and translocation. It was found that FtsH can initiate proteolysis of flavodoxin by translocating an internal loop to the protease chamber in an ATP dependent manner. We have also found that degradation of unfolded polypeptides by some chimeras of FtsH and its C.eleganshomologs do not require ATP hydrolysis. Firm evidence for the intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases has been obtained using mutants of the C.elegans fidgetin homolog, FIGL-1. We have also studied cellular functions of some AAA proteins in C.elegaas, which are homologous to causative factors of human genetic diseases. It was found that p97, which is related to inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, plays important roles in switching from spermatogenesis to oogenesis and progression of meiosis I as well as ERAD. We have observed that overexpression of p97 in C.elegans partially suppresses polyglutamine aggregate formation in vivo and that purified p97 suppresses aggregate formation of huntingtin fragments in an ATP-independent manner in vitro. We have shown spatiotemporally differential expression pattern of six UBX adaptor proteins of p97 in C.elegans. We showed that overexpression of SPAS-1, the C.elegans homolog of spastin related to hereditary spastic paraplegia, caused disassembly of microtubule network, indicating that SPAS-1 is involved in microtubule dynamics.