Modulating the fate of tau in the Hsp70 chaperone system.

描述(申请人提供)：本申请的目的是了解Hsp70的新型ATPase调节剂如何改变其病理上重要的客户蛋白tau的命运。中心假说指出，ATPase调节通过影响哪些辅助伴侣与Hsp70相关联来调节tau的命运。这项工作的完成将确定Hsp70复合体的命运，使tau进行折叠和降解。因此，新的和创新的方法，以药物干预阿尔茨海默病(AD)和其他牛皮病可能被识别。以下目标将检验中心假说。目的1：在细胞模型中鉴定Hsp70-ATPase调节剂诱导的复合体的辅伴侣组成。工作假说表明，Hsp70ATPase刺激物招募到一个辅助伴侣，促进tau的积累。相反，抑制剂招募辅助伴侣来促进tau蛋白酶体的降解。这一假设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验证。目的：研究ATPase调节剂对IHsp70与辅伴蛋白和客户蛋白tau相互作用的影响。工作模型假设Hsp70 ATPase调节改变了某些辅助伴侣与Hsp70-tau复合体的结合亲和力。将使用表面等离子体共振和酶联免疫吸附试验来测试ATPase调节剂对Hsp70、tau和辅助伴侣之间形成二元和三元复合体的影响。目的3：了解ATPase调控Hsp70、tau、共伴侣复合体形成的结构基础。我们预测，ATPase调节剂导致Hsp70主要填充特定的核苷酸结合构象。以前的工作，使用部分蛋白分解，已经表明独特的切割模式定义了Hsp70的ATP和ADP构象。因此，这项技术将被用来探测治疗后的Hsp70的结构。定点突变也将用于确定受ATPase调节剂影响的结合表面和变构网络。相关性：500多万美国人患有阿尔茨海默病(AD)。在阿尔茨海默病中，错误折叠的蛋白质会在大脑中积聚，导致严重的不可逆转的记忆丧失。通过治疗靶向参与错误折叠蛋白质折叠和降解的伴侣因子，我们可能能够利用人体自身的内在系统来治疗阿尔茨海默病。

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.