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.

项目摘要无处不在和保守的分子伴侣HSP70S和HSP110s构成了最重要的之一伴侣机器维持蛋白质稳态（蛋白质稳定）。但是，分子机制 HSP110在该伴侣机械中的确切作用在很大程度上不清楚。作为HSP70的遥远同源物， HSP110既是HSP70S的独立伴侣和副伴侣。作为独立的伴侣， HSP110s在防止变性蛋白的聚集时表现出独特的高伴侣活性，持有酶活性。作为辅助伴侣，HSP110S作为主要的核苷酸 - 交换因子（NEF）的功能 HSP70S。尽管NEF活性的重要性和机制是充分表征的，但功能和持有酶活性的参与几乎完全未知，这主要是由于缺乏任何可用的破坏这种持有酶活性的方法而不会影响NEF活性。 MSI3是唯一和必需的HSP110 在白色念珠菌中，是人类真菌感染最普遍的原因。我们的初步研究确定了一种新颖的MSI3抑制剂，名为2H。令人兴奋的是，2H专门废除了持有酶的活动使NEF活动在很大程度上完好无损。重要的是，看到2H可以减少体外和在体外折叠的蛋白质折叠 Vivo，提供了第一个直接证据，以支持HSP110持有酶活动的重要性 HSP70S/HSP110S伴侣机械。另外，作为真菌HSP110S的第一个抑制剂，有效2H 消除白色念珠菌在人类细胞中毒性有限的白色念珠菌的生长和生存能力，支持HSP110是设计用于真菌感染和2H的小说和有效疗法的重要目标可能代表未来药物化学工作的新型抗真菌剂的有前途的铅化合物。利用2H的唯一选择性，该提案的总体目标是两个方面：1）为了表征HSP110的HOLDASE活性的难以捉摸的分子机制和生物学功能， 2）增加对HSP110的机械理解，作为真菌感染的新治疗靶点。因此，我们提出了两个具体目标。 AIM 1：表征HSP110持有酶活动的体内功能在蛋白抑制中使用2H。利用酵母可用的功能强大且功能齐全的遗传学酿酒酵母，我们旨在直接分析HSP110S持有酶活性的保守功能蛋白抑制剂中的两个必不可少的体内过程：蛋白质折叠并进口到内质网。我们期望首次识别和表征HSP110的内源性底物。目标2：阐明抑制作用通过与MSI3复合物在复合物中的结构表征的机制。我们旨在解决MSI3的结构，单独并与2H复合，这将揭示2H和MSI3和MSI3和MSI3之间的特定结合相互作用 2H抑制MSI3的机制。我们预计我们的创新提议将使范式转移 HSP70S/HSP110S伴侣机械和PAVE中HSP110的功能和机制的发现我们将来通过靶向HSP110来开发新颖和选择性抗真菌的努力的坚实基础。