Structure and Regulation of hsp90 Chaperones

描述（由申请人提供）：hsp90伴侣参与各种细胞活动，包括从信号传导到细菌识别的蛋白质的后期成熟。细胞质Hsp90引导类固醇激素受体、原致癌激酶、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Hsp90抑制剂显示出有效的抗癌活性。GRP94，内质网hsp90类似物，伴侣蛋白的目的是运输到细胞表面和分泌，在从内质网输出毒性折叠蛋白中起关键作用，当靶向时导致肿瘤细胞死亡。hsp90伴侣蛋白的活性受ATP和其他配体与n端结构域结合的调控，该结构域的响应是hsp90伴侣蛋白周期的核心。然而，尽管它们具有高度的序列和结构同源性，细胞质和ER hsp90同源物在对调节配体的反应中表现出根本的差异。细胞质Hsp90的n端结构域盖的配体依赖性构象变化很难证明，并且尚未确定任何变化的合理触发因素。另一方面，在GRP94中，盖子上的一个保守插入启动了该区域进行构象重排，这取决于进入配体的身份。这些重排改变了伴侣的四级结构。本研究将利用x射线晶体学技术识别GRP94可获得的配体依赖构象状态，以可视化配体驱动GRP94配合物的结构。体内试验将用于评估这些结构重排的生物学重要性。GRP94对进入配体的敏感反应允许GRP94结合不能在细胞质Hsp90中容纳的抑制剂，因此对GRP94具有选择性。由于选择性配体和非选择性配体密切相关，我们将使用x射线晶体学来可视化结合GRP94和Hsp90的两类配体，并推断它们的选择性机制。由于每一种hsp90类似物都负责伴随不同的客户蛋白组，因此用选择性抑制剂特异性靶向一种hsp90类似物可能导致更高的疗效和治疗控制。最后，新的发展为表征与Hsp90和GRP94的新型客户端和共同伴侣相互作用提供了前景：最小的客户端蛋白装载系统为分离客户端-Hsp90复合物进行生化和生物物理研究开辟了道路，最近鉴定的第一个GRP94辅助因子os9首次为表征内质网平行中蛋白-蛋白相互作用的发生打开了大门。

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.