Molecular Chaperones and Small Molecules

描述（由申请人提供）：热休克蛋白70（Hsp 70）是一种分子伴侣，在蛋白质质量控制（PQC）中发挥核心作用。Hsp 70被认为是一种“分类伴侣”，因为它在蛋白质折叠中很重要，同时它也阻断聚集， 靶向错误折叠的蛋白质，通过泛素蛋白酶体系统（UPS）和分子伴侣介导的自噬（CMA）途径降解。Hsp 70如何决定蛋白质是否可以折叠？什么样的分子和结构机制将热休克蛋白70与其蛋白质底物的不同命运联系起来？这些问题对于我们理解细胞如何维持蛋白质稳态（即蛋白质稳态）至关重要。基于在第一个资助周期（2008年至今）中获得的发现，我们开发了一个模型，其中底物在Hsp 70复合物中的停留时间可能是影响蛋白质最终折叠或降解的一个因素。这个模型来自于我们使用高通量筛选（HTS）来发现新的化学探针的实验，这些化学探针可以“调节”Hsp 70的ATP酶活性。使用这些分子，我们发现抑制ATP周转有利于多种Hsp 70底物的降解，如tau和多聚谷氨酰胺（polyQ）扩增的亨廷顿蛋白（polyQ-Htt）和雄激素受体（polyQ-AR），而刺激活性导致底物积累和蛋白毒性。由于ATP营业额在热休克蛋白70的变构与底物亲和力，我们现在假设，长期与热休克蛋白70的相互作用可能是一个分子的“触发器”，有利于招聘UPS组件。在拟议的工作中，我们将探索这一想法在以下具体目标：（1）开发一套化学探针和点突变体，陷阱热休克蛋白70在其紧密或松散的亲和力形式，（2）探索底物亲和力的作用，在稳定的未折叠的模型蛋白，tau和polyQ-Htt，和（3）了解热休克蛋白70之间的区别正常的AR和错误折叠，polyQ-AR。通过这些研究，我们希望更好地了解Hsp 70如何做出关键的分诊决策。这项研究意义重大，因为Hsp 70与许多蛋白质错误折叠疾病有关，包括神经退行性疾病。因此，拟议的研究将阐明Hsp 70介导的PQC的逻辑，并可能提出治疗这些疾病的新方法。这项工作是创新的，因为我们将使用全面的化学生物学方法，将新的化学探针与点突变体和基于细胞的疾病模型相结合，以探索PQC的基本机制。

DESCRIPTION (provided by applicant): Heat shock protein 70 (Hsp70) is a molecular chaperone that plays a central role in protein quality control (PQC). Hsp70 is considered a "triage chaperone" because it is important in protein folding, while it also blocks aggregation and targets misfolded proteins for degradation by the ubiquitin proteasome system (UPS) and the chaperone-mediated autophagy (CMA) pathway. How does Hsp70 "decide" if a protein can be folded? What molecular and structural mechanisms link Hsp70 to the various fates of its protein substrates? These questions are essential to our understanding of how cells maintain protein homeostasis (i.e. proteostasis). Based on the findings obtained in the first funding cycle (2008-present), we have developed a model in which the dwell time of a substrate in the Hsp70 complex might be one factor that contributes to whether a protein is ultimately folded or degraded. This model emerged from experiments in which we used high throughput screening (HTS) to uncover new chemical probes that "tune" the ATPase activity of Hsp70. Using these molecules, we showed that inhibiting ATP turnover favored degradation of multiple Hsp70 substrates, such as tau and polyglutamine (polyQ) expanded huntingtin (polyQ-Htt) and androgen receptor (polyQ-AR), while stimulating activity led to substrate accumulation and proteotoxicity. Because ATP turnover in Hsp70 is allosterically linked to substrate affinity, we now hypothesize that prolonged interactions with Hsp70 may be a molecular "trigger" that favors recruitment of UPS components. In the proposed work, we will explore this idea in the following specific aims: (1) develop a suite of chemical probes and point mutants that trap Hsp70 in either its tight or loose affinity forms, (2) explore the role of substrate affinity in stabilization of te unfolded model proteins, tau and polyQ-Htt, and (3) understand how Hsp70 discriminates between normal AR and misfolded, polyQ-AR. From these studies, we expect to better understand how Hsp70 makes key triage decisions. This study is significant because Hsp70 has been linked to many protein-misfolding diseases, including neurodegenerative disorders. Thus, the proposed studies will clarify the logic of Hsp70-mediated PQC and, perhaps, suggest new methods for treating these diseases. This work is innovative because we will use a comprehensive chemical biology approach, combining new chemical probes with point mutants and cell-based models of disease to explore fundamental mechanisms of PQC.