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上重要的客户蛋白tau的命运。核心假说认为，atp酶调节通过影响与Hsp70相关的共伴侣来调节tau蛋白的命运。这项工作的完成将确定Hsp70复合物宿命tau的重折叠和降解。因此，可能会发现新的和创新的方法来药理学干预阿尔茨海默病（AD）和其他牛头病变。以下目标将检验中心假设。目的1：在细胞模型中鉴定由Hsp70 ATPase调节剂诱导的复合物的共伴侣组成。工作假设认为，Hsp70 atp酶刺激物招募了一种促进tau积累的共同伴侣。相反，抑制剂招募一种促进tau蛋白体降解的共伴侣。这一假设将通过在治疗和未治疗的细胞中进行tau免疫沉淀，然后进行质谱分析来验证。目的2：确定ATPase调节剂对IHsp70与共伴侣和客户蛋白tau结合相互作用的影响。工作模型假设Hsp70 ATPase调节改变了某些共伴侣对Hsp70-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试验将用于测试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。目的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ATPase调节剂导致Hsp70主要填充特定的核苷酸结合构象。先前的工作，利用部分蛋白水解，已经表明不同的切割模式定义了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。位点定向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）。在阿尔茨海默症中，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。通过治疗靶向参与折叠和降解错误折叠蛋白质的伴侣因子，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.