Molecular, cellular, and developmental studies on AAA chaperones

研究了AAA伴侣蛋白的ATP水解和底物蛋白易位等常见活性的分子机制。我们提出了AAA ATP酶在亚基间催化ATP水解的机制和底物易位的线程模型，并获得了这些模型的支持证据。AAA蛋白酶FtsH六聚体atp酶环孔处保守残基的突变表明，保守的芳香残基可能参与底物结合和易位。研究发现，FtsH可以通过ATP依赖的方式将一个内部环转移到蛋白酶室，从而启动黄氧多素的蛋白水解。我们还发现，一些FtsH嵌合体及其秀丽隐杆线虫同源物降解未折叠多肽不需要ATP水解。利用秀丽隐杆线虫的fidgetin同源突变体获得了AAA ATP酶在亚基间催化ATP水解机制的确凿证据，如图1所示。我们还研究了秀丽隐杆线虫中一些AAA蛋白的细胞功能，这些蛋白与人类遗传病的致病因子同源。我们发现p97与骨Paget病相关的包体肌病和额颞叶痴呆相关，在精子发生到卵子发生的转换和减数分裂I的进展以及ERAD中起重要作用。我们观察到秀丽隐杆线虫中p97的过表达在体内部分抑制聚谷氨酰胺聚集体的形成，纯化的p97在体外以atp不依赖的方式抑制亨廷顿蛋白片段的聚集体形成。我们发现了6种UBX接子蛋白p97在秀丽隐杆线虫中的时空差异表达模式。我们发现，与遗传性痉挛性截瘫相关的线虫的SPAS-1的过表达导致微管网络的破坏，这表明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.