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Hsp 70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。核心假设指出，ATP酶调节通过影响与Hsp 70相关的辅助分子伴侣介导tau的命运。这项工作的完成将确定Hsp 70复合物的命运tau的重折叠和降解。因此，可以确定新的和创新的方法来干预阿尔茨海默病（AD）和其他tau蛋白病。以下目标将检验中心假设。目的1：在基于细胞的模型中鉴定由Hsp 70 ATP酶调节剂诱导的复合物的共伴侣组成。工作假说指出，Hsp 70 ATP酶刺激剂募集促进tau积累的共伴侣。相反，抑制剂募集促进tau蛋白体降解的共分子伴侣。将通过在处理和未处理的细胞中进行tau的免疫沉淀，然后进行质谱分析来测试该假设。目的2：确定ATP酶调节剂对IHsp 70与辅伴侣和客户蛋白tau结合相互作用的影响。该工作模型假设Hsp 70 ATP酶调节改变了某些辅助分子伴侣对Hsp 70-tau复合物的结合亲和力。表面等离子体共振和酶联免疫吸附测定将用于测试ATP酶调节剂对热休克蛋白70，tau蛋白和辅分子伴侣之间的二元和三元复合物形成的影响。目的3：了解ATP酶调节对Hsp 70、tau蛋白、辅伴侣复合物形成的影响的结构基础。我们预测，ATP酶调节剂导致热休克蛋白70主要填充特定的核苷酸结合构象。以前的工作，使用部分蛋白水解，已经表明，独特的切割模式定义的ATP和ADP的热休克蛋白70的构象。因此，该技术将用于探测处理后的Hsp 70的结构。定点诱变也将用于定义受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，导致严重和不可逆的记忆丧失。通过治疗靶向参与错误折叠蛋白质的折叠和降解的伴侣因子，我们可能能够利用身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.