Structure and Regulation of hsp90 Chaperones

描述（由申请人提供）：hsp 90分子伴侣参与蛋白质的后期成熟，所述蛋白质涉及从信号传导到细菌识别的多种细胞活性。细胞质Hsp 90指导类固醇激素受体、原癌激酶、G蛋白和其他肿瘤转化的关键介质的成熟。破坏这种成熟的Hsp 90抑制剂显示出有效的抗癌活性。GRP 94是ER hsp 90的伴侣蛋白，其目的是运输到细胞表面并分泌，在从ER输出毒性错误折叠蛋白中起关键作用，并且当靶向时导致肿瘤细胞死亡。hsp 90分子伴侣的活性通过ATP和其他配体与N-末端结构域的结合来调节，并且该结构域的反应是hsp 90分子伴侣循环的中心。然而，尽管其高度的序列和结构同源性，细胞质和ER hsp 90旁系同源物表现出根本的差异，在他们的反应，调节配体。胞质Hsp 90的N-末端结构域盖子中的配体依赖性构象变化一直难以证明，并且尚未确定任何变化的合理触发因素。另一方面，在GRP 94中，盖中的保守插入使该区域经历构象重排，该构象重排根据引入配体的身份而显著不同。这些重排改变了分子伴侣的四级结构。本提案中的研究将使用X射线晶体学来识别GRP 94可用的配体依赖性构象状态，以可视化配体驱动的GRP 94复合物的结构。体内试验将用于评估这些结构重排的生物学重要性。GRP 94盖对进入的配体的敏感反应允许GRP 94结合不能容纳在细胞质Hsp 90中的抑制剂，因此对GRP 94具有选择性。由于选择性和非选择性配体是密切相关的，我们将使用X射线晶体学来可视化结合到GRP 94和Hsp 90的两类配体，并推断它们的选择性机制。因为每个hsp 90旁系同源物负责陪伴不同的客户蛋白组，所以用选择性抑制剂特异性靶向一个hsp 90旁系同源物可以导致更高的功效和治疗控制。最后，新的发展提供了表征新的客户端和与Hsp 90和GRP 94的共伴侣相互作用的前景：最小客户蛋白加载系统为分离客户-Hsp 90复合物以用于生物化学和生物物理学研究开辟了道路，并且最近对第一个GRP 94辅助因子os 9的鉴定，第一次，打开了表征蛋白质-蛋白质相互作用如何在内质网中发生的大门。

DESCRIPTION (provided by applicant): The hsp90 chaperones participate in the late stage maturation of proteins involved in diverse cellular activities ranging from signaling to bacterial recognition. Cytoplasmic Hsp90 guides the maturation of steroid hormone receptors, proto-oncogenic kinases, G-proteins, and other key mediators of neoplastic transformation. Inhibitors of Hsp90 that disrupt this maturation display potent anti-cancer activity. GRP94, the ER hsp90 paralog, chaperones proteins destined for transport to the cell surface and secretion, plays a key role in the export of toxic malfolded proteins from the ER, and when targeted leads to the death of tumor cells. The activity of hsp90 chaperones is regulated by the binding of ATP and other ligands to the N-terminal domain, and the response of this domain is central to the hsp90 chaperone cycle. Yet despite their high degree of sequence and structural homology, the cytoplasmic and ER hsp90 paralogs exhibit fundamental differences in their response to regulatory ligands. Ligand dependent conformational changes in the N-terminal domain lid of cytoplasmic Hsp90 have been difficult to demonstrate, and a plausible trigger for any changes has not been identified. In GRP94, on the other hand, a conserved insertion in the lid primes this region to undergo conformational rearrangements that differ dramatically depending on the identity of the incoming ligand. These rearrangements alter the quaternary structure of the chaperone. The research in this proposal will identify the ligand dependent conformational states available to GRP94 using X-ray crystallography to visualize the structures of ligand-driven GRP94 complexes. In vivo assays will be used to assess the biological importance of these structural rearrangements. The sensitive response of the GRP94 lid to incoming ligands allows GRP94 to bind inhibitors that cannot be accommodated in cytoplasmic Hsp90 and are therefore selective for GRP94. Because selective and non-selective ligands are closely related, we will use X-ray crystallography to visualize the both classes of ligands bound to GRP94 and Hsp90 and infer their mechanism of selectivity. Because each of the hsp90 paralogs is responsible for chaperoning distinct sets of client proteins, specific targeting of one hsp90 paralog with selective inhibitors may result in higher efficacy and therapeutic control. Finally, new developments offer the prospect of characterizing novel client and co-chaperone interactions with both Hsp90 and GRP94: minimal client protein loading systems open the way to isolating client-Hsp90 complexes for biochemical and biophysical study, and the recent identification of the first GRP94 accessory factor, os9, has, for the first time, opened the door to characterizing how protein-protein interactions occur in the ER paralog.