Post-translational modifications of Hsp90 that impact drug efficacy

热休克蛋白90（Heat shock protein 90，Hsp 90）是一种分子伴侣，它是许多信号蛋白稳定和功能所必需的，这些信号蛋白通常在癌细胞中被激活、突变或过表达，并且是癌细胞增殖和存活的基础。热休克蛋白90是一种构象灵活的蛋白质，它与一组不同的辅分子伴侣相关联，这取决于氨基末端结合口袋的ATP或ADP占用率。Hsp 90自身的核苷酸交换和ATP水解，在辅分子伴侣的协助下，驱动Hsp 90分子伴侣机器结合、分子伴侣和释放客户蛋白。Hsp 90分子伴侣机器的循环对其功能至关重要。虽然ATP结合和水解已经令人信服地参与调节Hsp 90循环，越来越多的证据表明，Hsp 90的各种翻译后修饰，包括磷酸化，乙酰化，sumoylation和其他修饰，提供了额外的重叠或平行水平的调节。更全面地了解这些不同的蛋白质修饰是如何在细胞水平上调节和相互作用以调节Hsp 90分子伴侣活性的，这对于设计抑制这种医学上重要的分子靶点的新方法至关重要。调节Hsp 90的不同翻译后修饰的信号通路的协调是非常可能的。理解各种修饰之间的相互作用无疑是一项艰巨的任务，但这些知识将大大增加我们对Hsp 90功能在细胞复杂环境中如何调节的理解。这些信息可能提供一种独特的方法，在癌细胞中特异性阻断Hsp 90的功能，因此将是一个重要的考虑因素，在设计Hsp 90抑制剂与其他分子靶向药物的临床试验。对翻译后修饰在调节Hsp 90功能中所起作用的更透彻理解肯定会提高此类联合治疗的有效性。在Fy 14中，我们发现两种化学上不相关的Hsp 90抑制剂，苯醌安莎霉素格尔德霉素和嘌呤类似物PU-H71，选择重叠但不相同的总细胞Hsp 90亚群，即使这两种抑制剂结合到氨基末端核苷酸口袋，并防止N结构域二聚化。我们的数据还表明，PU-H71能够获得比格尔德霉素更广泛的N结构域未二聚化的Hsp 90构象，并且受Hsp 90磷酸化的影响较小，这与其更广泛和更有效的抗肿瘤活性一致。更全面地了解细胞环境对小分子抑制剂与Hsp 90结合的影响，将有助于它们在临床上更有效地使用。此外，我们发现，不对称的Hsp 90 N结构域suMO化招募的辅助分子伴侣Aha 1和ATP竞争性抑制剂。热休克蛋白90介导的ATP水解需要一系列的构象变化，这些变化受辅伴侣和许多翻译后修饰（PTM）的调节。SUMO化是最不了解的Hsp 90 PTM之一。我们发现酵母（K178）和人（K191）Hsp 90的N结构域中保守赖氨酸残基的不对称SUMO化促进ATP酶激活辅伴侣蛋白Aha 1的募集，并且出乎意料地，也有利于Hsp 90抑制剂的结合，这表明这些药物优先与积极参与伴侣循环的Hsp 90蛋白结合。重要的是，细胞转化伴随着稳态N结构域SUMO化的升高，并且增加的Hsp 90 SUMO化使酵母和哺乳动物细胞对Hsp 90抑制剂敏感，提供了解释癌细胞对这些药物敏感性的机制。

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of many signaling proteins that are often activated, mutated or over-expressed in cancer cells and that underly cancer cell proliferation and survival. Hsp90 is a conformationally flexible protein that associates with a distinct set of co-chaperones depending on ATP or ADP occupancy of an amino-terminal binding pocket. Nucleotide exchange and ATP hydrolysis by Hsp90 itself, with the assistance of co-chaperones, drive the Hsp90 chaperone machine to bind, chaperone, and release client proteins. Cycling of the Hsp90 chaperone machine is critical to its function. Although ATP binding and hydrolysis have been convincingly implicated in regulating the Hsp90 cycle, growing evidence suggests that various post-translational modifications of Hsp90, including phosphorylation, acetylation, sumoylation and other modifications, provide an additional overlapping or parallel level of regulation. A more complete understanding of how these various protein modifications are regulated and interact with each other at the cellular level to modulate Hsp90 chaperone activity is critical to the design of novel approaches to inhibit this medically important molecular target. Coordination of signaling pathways that mediate distinct post-translational modifications of Hsp90 is highly likely. Understanding the cross-talk between various modifications will no doubt be a difficult undertaking, but such knowledge will add greatly to our appreciation of how Hsp90 function is regulated in the complex milieu of the cell. Such information may provide a unique approach to specific interdiction of Hsp90 function in cancer cells and will thus be an important consideration in designing clinical trials of Hsp90 inhibitors in combination with other molecularly targeted drugs. A more thorough understanding of the role that post-translational modifications play in modulating Hsp90 function will certainly improve the effectiveness of such combination therapies. In Fy14, we found that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic. Further, we found that asymmetric Hsp90 N domain sUMOylation recruits the co-chaperone Aha1 and ATP-competitive inhibitors. Hsp90-mediated ATP hydrolysis requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs.We found that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the ATPase-activating cochaperone Aha1 and, unexpectedly, also favors the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.