Post-translational modifications of Hsp90 that impact drug efficacy

热休克蛋白90(Hsp90)是一种分子伴侣，在癌细胞中经常被激活、突变或过度表达，影响癌细胞的增殖和存活，是许多信号蛋白稳定和发挥功能所必需的。HSP90是一种构象灵活的蛋白质，它与一组不同的辅助伴侣结合，这取决于氨基末端结合口袋的ATP或ADP占有率。Hsp90自身的核苷酸交换和ATP水解，在辅助伴侣的帮助下，驱动Hsp90伴侣机器结合、伴侣和释放客户蛋白。Hsp90伴侣机器的循环对其功能至关重要。尽管ATP结合和水解被认为与Hsp90循环的调控有关，但越来越多的证据表明，Hsp90的各种翻译后修饰，包括磷酸化、乙酰化、和甲基化和其他修饰，提供了额外的重叠或平行的调节水平。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并相互作用以调节Hsp90伴侣活性的，对于设计新的方法来抑制这一医学上重要的分子靶点至关重要。调节Hsp90不同翻译后修饰的信号通路的协调是很有可能的。理解不同修饰之间的相互作用无疑是一项困难的工作，但这些知识将极大地增加我们对Hsp90功能如何在复杂的细胞环境中进行调控的理解。这些信息可能提供一种独特的方法来特异性阻断癌细胞中的Hsp90功能，因此将是设计Hsp90抑制剂与其他分子靶向药物联合临床试验的重要考虑因素。更透彻地了解翻译后修饰在调节Hsp90功能中所起的作用，肯定会提高这种联合疗法的有效性。在2014财年，我们发现两种化学上不相关的Hsp90抑制剂，苯醌阿萨霉素和嘌呤类似物PU-H71，选择总细胞Hsp90的重叠但不相同的亚群，即使这两种抑制剂都与氨基末端核苷酸口袋结合，并防止N结构域二聚。我们的数据还表明，PU-H71能够获得比格尔达霉素更广泛的N结构域非二聚体Hsp90构象，并且受Hsp90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Hsp90结合的影响，将有助于它们在临床上更有效地使用。此外，我们还发现，不对称的Hsp90N结构域求和作用招募了辅助伴侣AHA1和ATP竞争性抑制物。HSP90介导的ATP水解需要一系列的构象变化，这些变化由辅伴侣和大量的翻译后修饰(PTM)调节。SUMO化是人们最不了解的Hsp90 PTM之一。我们发现，酵母(K178)和人(K191)Hsp90 N域保守赖氨酸残基的不对称SUMO化既促进了激活ATPase的辅伴侣Aha1的招募，又出人意料地有利于Hsp90抑制剂的结合，表明这些药物优先与活跃在伴侣环中的Hsp90蛋白结合。重要的是，细胞转化伴随着稳态N结构域SUMO化的增加，而Hsp90 SUMO化的增加使酵母和哺乳动物细胞对Hsp90抑制剂敏感，这为解释癌细胞对这些药物的敏感性提供了一种机制。

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.