Structural and functional studies of Hsp70/Hsp110 molecular chaperones

项目摘要 普遍存在和保守的分子伴侣Hsp 70和Hsp 110形成了一个最重要的 蛋白质稳态（proteostasis）的分子伴侣机制。然而，分子机制 Hsp 110在这种分子伴侣机制中的确切作用仍不清楚。作为热休克蛋白70的远缘同源物， Hsp 110是Hsp 70的独立分子伴侣和共分子伴侣。作为独立的监护人， 热休克蛋白110在防止变性蛋白聚集方面表现出独特的高分子伴侣活性， 保持酶活性作为辅助分子伴侣，Hsp 110 s作为主要的核苷酸交换因子（NEF）， HSP 70虽然NEF活性的重要性和机制已得到充分表征，但其功能和作用机制仍有待进一步研究。 参与保持酶活性几乎完全未知，主要是由于缺乏任何可用的 方法来破坏这种保持酶活性而不影响NEF活性。Msi 3是Hsp 110中唯一和必需的 白色念珠菌是人类真菌感染的最常见原因。我们的初步研究表明 发现了一种新的Msi 3抑制剂，命名为2 H。令人兴奋的是，2 H特异性地消除保持酶活性， 而NEF的活性基本上保持不变。重要的是，2 H在体外和体内都能减少蛋白质折叠。 体内，提供了第一个直接证据，以支持Hsp 110的保持酶活性的重要性， Hsp 70 s/Hsp 110 s伴侣机器。此外，2 H作为真菌Hsp 110 s的第一个抑制剂， 消除了C.白色念珠菌在人类细胞中的毒性有限，支持Hsp 110是 2 H是设计新型和有效的真菌感染治疗药物的一个重要靶点，2 H可能代表了一种新的治疗方法。 有前途的先导化合物为一类新的抗真菌药物，为未来的药物化学的努力。 利用2 H的独特选择性，该提案的总体目标是双重的：1） 表征Hsp 110 s保持酶活性的难以捉摸的分子机制和生物学功能， 2)增加对Hsp 110作为真菌感染的新治疗靶点的机制理解。 因此，我们提出两个具体目标。目的1：研究热休克蛋白110保持酶活性的体内功能 在蛋白质稳态中使用2 H。利用酵母强大而简单的遗传特性 在酿酒酵母中，我们的目的是直接分析Hsp 110 s的保持酶活性在酿酒酵母中的保守功能。 蛋白质稳态中的两个基本体内过程：蛋白质折叠和输入内质网。我们预计 首次鉴定和表征Hsp 110的内源性底物。目的2：阐明抑制性 通过与Msi 3复合的2 H的结构表征的机制。我们的目标是解决Msi 3的结构， 单独和与2 H复合，这将揭示2 H和Msi 3之间的特异性结合相互作用， 2 H对Msi 3.我们希望我们的创新提案将使范式转变 Hsp 110在Hsp 70/Hsp 110分子伴侣机制中的作用和机制的发现 这为我们未来通过靶向Hsp 110开发新型和选择性抗真菌药物奠定了坚实的基础。

Project Summary Ubiquitous and conserved molecular chaperones Hsp70s and Hsp110s form one of the most essential chaperone machineries in maintaining protein homeostasis (proteostasis). However, the molecular mechanisms and exact role of Hsp110s in this chaperone machinery remain largely unclear. As distant homologs of Hsp70s, Hsp110s are both independent chaperones and co-chaperones for Hsp70s. As independent chaperones, Hsp110s demonstrate a unique high chaperone activity in preventing aggregation of denatured proteins, the holdase activity. As co-chaperones, Hsp110s function as the major nucleotide-exchange factor (NEF) for Hsp70s. Although the importance and mechanism of the NEF activity is well-characterized, the function and involvement of the holdase activity is almost completely unknown primarily due to the lack of any available approach to disrupt this holdase activity without affecting the NEF activity. Msi3 is the sole and essential Hsp110 in Candida albicans, the most prevalent cause of fungal infections in humans. Our preliminary studies have identified a novel inhibitor for Msi3, named 2H. Excitingly, 2H specifically abolishes the holdase activity while leaving the NEF activity largely intact. Importantly, 2H was seen to reduce protein folding both in vitro and in vivo, providing the first direct evidence to support the importance of the holdase activity of an Hsp110 in the Hsp70s/Hsp110s chaperone machinery. In addition, as the first inhibitor for fungal Hsp110s, 2H effectively eliminates the growth and viability of C. albicans with limited toxicity in human cells, supporting that Hsp110s are an important target for designing novel and potent therapeutics for fungal infections and 2H may represent a promising lead compound for a new class of antifungals for future medicinal chemistry efforts. Taking advantage of the unique selectivity of 2H, the overall objective of this proposal is two-fold: 1) to characterize the elusive molecular mechanism and biological function of the holdase activity of Hsp110s, and 2) to increase mechanistic understanding of Hsp110s as a new therapeutic target for fungal infections. Accordingly, we propose two Specific Aims. Aim 1: Characterize the in vivo function of Hsp110's holdase activity in proteostasis using 2H. Taking advantage of the powerful and facile genetics available to the yeast Saccharomyces cerevisiae, we aim to directly analyze the conserved functions of Hsp110s' holdase activity in two essential in vivo processes in proteostasis: protein folding and import into endoplasmic reticulum. We expect to identify and characterize endogenous substrates for Hsp110s for the first time. Aim 2: Elucidate the inhibitory mechanism via structural characterization of 2H in complex with Msi3. We aim to solve the structures of Msi3, both alone and complexed with 2H, which will reveal the specific binding interactions between 2H and Msi3 and the mechanism of 2H's inhibition of Msi3. We expect that our innovative proposal will make paradigm-shifting discoveries on the function and mechanism of Hsp110s in the Hsp70s/Hsp110s chaperone machinery and pave a solid foundation for our future effort to develop novel and selective antifungals by targeting Hsp110s.