Modulating the fate of tau in the Hsp70 chaperone system.

描述（由申请人提供）：本申请的目的是了解 Hsp70 的新型 ATP 酶调节剂如何改变其病理学上重要的客户蛋白 tau 的命运。中心假设指出，ATP 酶调节通过影响与 Hsp70 相关的共伴侣来介导 tau 的命运。这项工作的完成将确定决定 tau 重折叠和降解的 Hsp70 复合物。因此，可以确定药物干预阿尔茨海默氏病（AD）和其他 tau蛋白病的新的和创新的方法。以下目标将检验中心假设。目标 1：在细胞模型中鉴定 Hsp70 ATP 酶调节剂诱导的复合物的共伴侣组成。工作假设指出，Hsp70 ATP 酶刺激剂会招募辅助伴侣，促进 tau 蛋白的积累。相反，抑制剂招募辅助伴侣，促进 tau 蛋白体降解。该假设将通过在处理和未处理的细胞中进行 tau 蛋白免疫沉淀，然后进行质谱分析来检验。目标 2：确定 ATP 酶调节剂对 IHsp70 与辅助伴侣和客户蛋白 tau 结合相互作用的影响。该工作模型假设 Hsp70 ATP 酶调节改变了某些辅助伴侣与 Hsp70-tau 复合物的结合亲和力。表面等离子共振和酶联免疫吸附测定将用于测试 ATP 酶调节剂对 Hsp70、tau 和共伴侣之间二元和三元复合物形成的影响。目标 3：了解 ATP 酶调节对 Hsp70、tau、共伴侣复合物形成影响的结构基础。我们预测 ATP 酶调节剂会导致 Hsp70 主要填充特定的核苷酸结合构象。之前的工作使用部分蛋白水解，表明独特的切割模式定义了 Hsp70 的 ATP 和 ADP 构象。因此，该技术将用于探测处理后的Hsp70的结构。定点诱变还将用于定义受 ATP 酶调节剂影响的结合表面和变构网络。相关性：超过 500 万美国人患有阿尔茨海默病 (AD)。在 AD 中，错误折叠的蛋白质在大脑中积聚，导致严重且不可逆转的记忆丧失。通过治疗性靶向参与错误折叠蛋白质折叠和降解的伴侣因子，我们也许能够利用人体自身的内在系统来治疗 AD。

DESCRIPTION (provided by applicant): The objective of this application is to understand how novel ATPase modulators of Hsp70 change the fate of its pathologically important client protein, tau. The central hypothesis states that ATPase modulation mediates the fate of tau by affecting which co-chaperones associate with Hsp70. Completion of this work will define the Hsp70 complexes that fate tau for refolding and degradation. Consequently, new and innovative ways to pharmacologically intervene in Alzheimer's disease (AD) and other tauopathies may be identified. The following aims will test the central hypothesis. Aim 1: Identify the co-chaperone composition of complexes induced by Hsp70 ATPase modulators in a cell- based model. The working hypothesis states that Hsp70 ATPase stimulators recruit a co- chaperone which promotes the accumulation of tau. Conversely, Inhibitors recruit a co-chaperone which promotes proteosomal degradation of tau. This hypothesis will be tested by performing an immunoprecipitatlon of tau, in both treated and untreated cells, followed by mass spectrometry. Aim 2: Determine the effect of ATPase modulators on IHsp70 binding interactions with co- chaperones and client protein tau. The working model postulates that Hsp70 ATPase modulation changes the binding affinity of certain co-chaperones for the Hsp70-tau complex. Surface plasmon resonance and an enzyme-linked immunosorbent assay will be used to test the effect of ATPase modulators on the formation of binary and ternary complexes between Hsp70, tau, and co-chaperones. Aim 3: Understand the structural basis for the effect of ATPase modulation on Hsp70, tau, co- chaperone complex formation. We predict that ATPase modulators cause Hsp70 to predominantly populate specific nucleotide bound conformations. Previous work, using partial proteolysis, has shown that distinctive cleavage patterns define the ATP and ADP conformations of Hsp70. Thus, this technique will be used to probe the structure of Hsp70 after treatment. Site-directed mutagenesis will also be used to define binding surfaces and allosteric networks affected by ATPase modulators. Relevance: More than five million Americans suffer from Alzheimer's disease (AD). In AD, misfolded proteins build up in the brain causing severe and irreversible memory loss. By therapeutically targeting a chaperone factor involved in the folding and degradation of misfolded proteins we may be able to take advantage of the body's own intrinsic system to treat AD.