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Structural and functional studies of Hsp70/Hsp110 molecular chaperones

Hsp70/Hsp110分子伴侣的结构和功能研究

基本信息

批准号：
10753661
负责人：
Qinglian Liu
金额：
$ 38.22万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2027-06-30
项目状态：
未结题

项目摘要

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.

项目概要普遍存在的保守分子伴侣 Hsp70 和 Hsp110 是最重要的分子伴侣之一维持蛋白质稳态（蛋白质稳态）的伴侣机制。然而，分子机制 Hsp110 在这种伴侣机制中的确切作用仍不清楚。作为 Hsp70 的远距离同系物， Hsp110 既是 Hsp70 的独立伴侣，又是辅助伴侣。作为独立监护人， Hsp110s 在防止变性蛋白质聚集方面表现出独特的高伴侣活性，保持酶活性。作为共伴侣，Hsp110 充当主要核苷酸交换因子 (NEF) 热休克蛋白70s。尽管 NEF 活动的重要性和机制已得到充分表征，但其功能和保持酶活性的参与几乎完全未知，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。 Msi3 是唯一且重要的 Hsp110 白色念珠菌是人类真菌感染最常见的原因。我们的初步研究有鉴定出一种新型 Msi3 抑制剂，命名为 2H。令人兴奋的是，2H 特异性地消除了保持酶活性，同时 NEF 活动基本完好无损。重要的是，2H 在体外和体内都被认为可以减少蛋白质折叠。体内，提供了第一个直接证据来支持 Hsp110 的保持酶活性在 Hsp70s/Hsp110s 伴侣机械。此外，作为第一个真菌Hsp110s抑制剂，2H可以有效消除了白色念珠菌的生长和活力，对人体细胞的毒性有限，支持 Hsp110 是设计针对真菌感染的新型有效疗法的重要目标，2H 可能代表有望成为未来药物化学工作中新型抗真菌药物的先导化合物。利用 2H 的独特选择性，该提案的总体目标有两个：1) 表征 Hsp110 的保持酶活性难以捉摸的分子机制和生物学功能，以及 2）增加对Hsp110作为真菌感染新治疗靶点的机制理解。因此，我们提出两个具体目标。目标 1：表征 Hsp110 保持酶活性的体内功能使用 2H 进行蛋白质稳态。利用酵母强大而简单的遗传学优势酿酒酵母，我们的目的是直接分析 Hsp110s 保持酶活性的保守功能蛋白质稳态的两个重要体内过程：蛋白质折叠和导入内质网。我们期望首次鉴定和表征 Hsp110 的内源底物。目标 2：阐明抑制因素通过 2H 与 Msi3 复合物的结构表征来了解其机制。我们的目标是解决Msi3的结构，单独和与 2H 复合，这将揭示 2H 和 Msi3 之间的特异性结合相互作用 2H抑制Msi3的机制。我们期望我们的创新提案将带来范式转变 Hsp70s/Hsp110s分子伴侣机制中Hsp110s功能和机制的发现及铺路为我们未来通过针对 Hsp110 开发新型选择性抗真菌药物奠定了坚实的基础。