Elucidating the Mechanistic Details of the Grp94 Molecular Chaperone through an Integrated Computational and Experimental Approach

项目总结/摘要 伴侣蛋白对细胞存活至关重要，并为细胞内正确的蛋白质折叠提供途径。 我们的研究重点是内质网中的ATP依赖性伴侣，特别是Grp 94。GRP94 属于高度保守的Hsp 90超家族，与其胞质和线粒体旁系同源物一样， 并激活特定的客户蛋白。Grp 94是基本的兴趣，因为它具有有限的机械特性， 与其他paralogs相比。Grp 94也是独特的，因为它具有几个观察到的结构和特性。 与旁系同源物的功能差异使其成为有前途的药物靶点。尽管Grp 94的重要性 虽然在蛋白质稳态中存在伴侣，但其伴侣循环的基本细节还没有很好地表征。 GRP 94具有实际意义，因为内质网中异常的蛋白质折叠导致医学上的疾病。 2型糖尿病、癌症、B & C型肝炎、神经退行性疾病和心血管疾病等问题。 了解Grp 94的结构和功能将为了解疾病如何引起 错误折叠的ER蛋白可以被治疗和预防。Grp 94机制的基本知识 可以帮助合理的药物设计。目前的策略涉及靶向所有Hsp 90的ATP竞争性抑制剂 旁系同源物并抑制生产性和非生产性伴侣活性，这导致毒性。小说 治疗策略包括抑制毒性蛋白质的陪伴，同时保留无毒蛋白质的陪伴。 蛋白质和靶向特定的Hsp 90旁系同源物;然而，需要机制信息来移动场 朝这个方向因此，对这种分子伴侣机制的研究将具有巨大的实用价值。 在疾病治疗的发展中具有经济价值。了解构象变化 参与伴侣循环的人对于确定干预点至关重要。理解 客户蛋白和其他分子伴侣在结构和功能上如何相互作用对于 设计Grp 94的竞争性调节剂。 我的实验室将开发新技术用于Grp 94的功能和结构研究。我们将紧紧地结合在一起 实验和计算研究，这是一个强大的工具组合，将使我们能够 阐明单独使用任何一种方法都不可能获得的分子细节。使用此 我们将回答以下相关的生物学问题：（1）什么是首选的 Grp 94的构象状态，以及哪些构象在平衡中共存？(2)细胞状况如何 和相互作用影响Grp 94的构象采样？(3)Grp 94在细胞内的哪种分子伴侣活性 演示和要求是什么？(4)客户蛋白在Grp 94上的何处相互作用？(5)是ATP 水解事件中Grp 94二聚体对称或不对称？这些研究的成功完成是 预期在去卷积Grp 94分子伴侣机制中具有重要影响， 所涉及的变化，以及底物蛋白相互作用和加工的细节。这些信息将 有助于发现新的和新颖的干预点，以改善疾病。

Project Summary/Abstract Chaperone proteins are critical for cell survival and provide pathways for correct protein folding within the cell. Our research focuses on ATP-dependent chaperones in the endoplasmic reticulum, specifically Grp94. Grp94 belongs to the highly conserved Hsp90 superfamily, and like its cytosolic and mitochondrial paralogs, it folds and activates specific client proteins. Grp94 is of fundamental interest because it has limited mechanistic information compared to other paralogs. Grp94 is also unique because it has several observed structural and functional differences from paralogs that make it a promising drug target. Despite the importance of Grp94 chaperoning in protein homeostasis, the fundamental details of its chaperone cycle are not well characterized. Grp94 is of practical interest since aberrant protein folding in the endoplasmic reticulum results in medical issues such as type 2 diabetes, cancer, hepatitis B & C, and neurodegenerative and cardiovascular diseases. Understanding Grp94’s structure and function will provide a foundation for understanding how diseases caused by misfolded ER proteins can be treated and prevented. This fundamental knowledge of Grp94 mechanisms can aid in rational drug design. Current strategies involve ATP-competitive inhibitors that target all Hsp90 paralogs and inhibit both productive and unproductive chaperone activity, which results in toxicity. Novel therapeutic strategies include inhibiting chaperoning of toxic proteins while retaining chaperoning of non-toxic proteins and targeting specific Hsp90 paralogs; however, mechanistic information is required to move the field in this direction. Therefore, the studies of this chaperone mechanism will be of immense practical and economical value in the development of disease therapies. Understanding the conformational changes involved in the chaperone cycle is fundamentally important for identifying points of intervention. Understanding how client proteins and other chaperones structurally and functionally interact is fundamentally important for designing competitive modulators of Grp94. My lab will develop novel techniques for functional and structural studies of Grp94. We will tightly couple experimental and computational studies, which is a powerful combination of tools that will enable us to elucidate molecular details that wouldn’t be possible to obtain with either method individually. Using this approach, we will answer the following pertinent biological questions: (1) What are the preferred conformational states of Grp94 and which conformations co-exist in equilibrium? (2) How do cellular conditions and interactors influence Grp94’s conformational sampling? (3) What type of chaperone activity does Grp94 demonstrate and what are the requirements? (4) Where do client proteins interact on Grp94? (5) Are ATP hydrolysis events in the Grp94 dimer symmetric or asymmetric? The successful completion of these studies is expected to have an important impact in deconvoluting the Grp94 chaperone mechanism, the structural changes involved, and the details of substrate protein interactions and processing. This information will facilitate in finding new and novel points of intervention to ameliorate disease.