Post-translational modifications of Hsp90 that impact drug efficacy

热休克蛋白90 （Hsp90）是许多信号蛋白的稳定性和功能所必需的分子伴侣，这些信号蛋白在癌细胞中经常被激活、突变或过度表达，是癌细胞增殖和存活的基础。Hsp90是一种构象灵活的蛋白，根据ATP或ADP在氨基末端结合袋中的占用情况，与一组不同的共伴侣结合。Hsp90自身的核苷酸交换和ATP水解，在共伴侣的帮助下，驱动Hsp90伴侣机器结合、伴侣和释放客户蛋白。Hsp90伴侣机的循环对其功能至关重要。尽管ATP结合和水解已经令人信服地参与了Hsp90周期的调节，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被调节和相互作用以调节Hsp90伴侣活性的，对于设计抑制这一医学上重要的分子靶标的新方法至关重要。极有可能协调介导不同的Hsp90翻译后修饰的信号通路。了解各种修饰之间的串扰无疑是一项艰巨的任务，但这些知识将大大增加我们对Hsp90功能在细胞复杂环境中如何调节的认识。这些信息可能为特异性阻断Hsp90在癌细胞中的功能提供了一种独特的方法，因此将成为设计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。更深入地了解翻译后修饰在调节Hsp90功能中的作用，必将提高这类联合治疗的有效性。Hsp90受到一系列影响其功能的翻译后修饰，包括乙酰化。组蛋白去乙酰化酶（HDAC）抑制剂和HDAC6的敲低可诱导Hsp90乙酰化并抑制其活性。然而，直接确定Hsp90乙酰化的功能后果需要对特定位点进行定位。我们已经证明hsp90k294是乙酰化的。K294的突变分析表明，它的乙酰化状态是客户蛋白和伴侣蛋白结合的重要决定因素。在酵母中，与WT或模拟组成乙酰化的突变体相比，不能在K294位点乙酰化的Hsp90突变体的生存能力和伴侣蛋白功能降低。这些数据表明，K294的乙酰化/去乙酰化在调控Hsp90伴侣蛋白周期中起重要作用。进一步的研究集中在Hsp90中特定氨基酸的磷酸化，以及这些翻译后变化对Hsp90对抑制性药物、共伴侣和亚细胞定位的亲和力的影响。

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, 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. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We have demonstrated that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle. Further studies are focusing on phosphorylation of specific amino acids in Hsp90 and of the affect of these post-translational changes on Hsp90 affinity for inhibitory drugs, co-chaperones and subcellular localization.