Probing the Role of Chaperone-TPR Complexes in Tau Proteostasis

抽象的。一种无法治愈的神经退行性疾病，包括进行性 核上性麻痹（PSP）、皮质基底节变性（CBD）和阿尔茨海默病（AD）与 脑内神经纤维缠结（NFT）的积累。这些聚集体主要由 微管相关蛋白tau（MAPT/tau），其通过翻译后修饰而被大量修饰 （PTM）。如果我们了解了神经元通常是如何调节tau蛋白的PTM的，我们也许能够找到恢复tau蛋白的方法。 微妙的平衡。这个项目的目的是了解分子伴侣，热休克 蛋白70（Hsp 70）和热休克蛋白90（Hsp 90），直接修饰tau蛋白。Hsp 70和Hsp 90是已知的 来绑定tau中的关键易聚集位置。虽然这些相互作用提高了tau的溶解度，但它们也 招募了许多关键的PTM酶，包括：（i）E3泛素连接酶CHIP，（ii）顺-反脯氨酰 异构酶FKBP 51和（iii）蛋白磷酸酶PP 5。具体地说，这些酶中的每一种都含有一种 四肽重复（TPR）结构域，其对C-末端的保守EEVD-CO2 H基序具有亲和力 Hsp 70和Hsp 90因此，我们假设伴侣蛋白和TPR结构域之间的复合物最终 决定了tau蛋白的构象以及它是泛素化还是去磷酸化。由于创新， cryo-EM和最近在大规模肽库和抗体设计方面的突破，我们准备使用 结构和化学方法来理解伴侣蛋白如何与TPR蛋白协调来修饰tau。 在初步研究中，我们已经测量了伴侣的EEVD基序与一组TPR结构域的结合， 揭示了Hsp 70对PP 5和Hsp 90对FKBP 51的意想不到的选择性，以及Hsp 70对PP 5和FKBP 51的关键作用。 磷酸化调节这些亲和力。我们还创建了一个包含约640，000个肽序列的文库， 用它来鉴定一种有效的（<10 nM）CHIP TPR结构域抑制剂，该抑制剂不与密切相关的TPR结合。 最后，我们验证了研究Hsp 70-CHIP-tau三元复合物的结构方法， 它已经揭示了ADP的一个令人惊讶的重要作用。在这些令人兴奋的发现的指导下， 通过基于团队的方法，我们建议研究tau如何与：（SA 1）Hsp 70/Hsp 90-CHIP，（SA 2） Hsp 90-FKBP 51和（SA 3）Hsp 70-PP 5。通过这项工作，我们的目标是确定分子开关在 关键蛋白质界面调节tau的结构和修饰。我们预计这些研究也可以 揭示了治疗tau蛋白病的新药物靶点。

Abstract. A devastating class of untreatable, neurodegenerative disorders, which includes progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and Alzheimer’s disease (AD), are associated with accumulation of neurofibrillary tangles (NFTs) in the brain. These aggregates are primarily composed of microtubule-associated protein tau (MAPT/tau), which is heavily modified by post-translational modifications (PTMs). If we understood how neurons normally regulate tau’s PTMs, we might be able to find ways of restoring its delicate balance. The objective of this project is to understand how the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90), direct modifications on tau. Hsp70 and Hsp90 are known to bind key, aggregation-prone locations in tau. While these interactions improve the solubility of tau, they also recruit a number of critical PTM enzymes including: (i) the E3 ubiquitin ligase CHIP, (ii) the cis-trans prolyl isomerase FKBP51 and (iii) the protein phosphatase PP5. Specifically, each of these enzymes contains a tetratricopeptide repeat (TPR) domain, which has affinity for the conserved EEVD-CO2H motif at the C-termini of Hsp70 and Hsp90. Thus, we hypothesize that complexes between chaperones and TPR domains ultimately determines tau’s conformation and whether it is ubiquitinated or de-phosphorylated. Thanks to innovations in cryo-EM and recent breakthroughs in large-scale peptide libraries and antibody design, we are poised to use structural and chemical approaches to understand how chaperones coordinate with TPR proteins to modify tau. In preliminary studies, we have measured binding of the chaperone’s EEVD motifs to a panel of TPR domains, revealing unexpected selectivity of Hsp70 for PP5 and Hsp90 for FKBP51, as well as a key role for phosphorylation in tuning these affinities. We have also created a library of ~640,000 peptide sequences and used it to identify a potent (<10 nM) inhibitor of CHIP’s TPR domain that does not bind closely related TPRs. Finally, we have validated a structural approach to studying the Hsp70-CHIP-tau ternary complex, a project which has already uncovered a surprising, essential role for ADP. Guided by these exciting findings and fueled by a team-based approach, we propose to study how tau interacts with: (SA1) Hsp70/Hsp90-CHIP, (SA2) Hsp90-FKBP51 and (SA3) Hsp70-PP5. Through this effort, we aim to determine how molecular switches at the key protein interfaces tune the structure and modifications on tau. We anticipate that these studies could also revealing new drug targets for treating tauopathies.