Probing the Role of Chaperone-TPR Complexes in Tau Proteostasis

抽象的。一类毁灭性的无法治疗的神经退行性疾病，包括进行性的 核上性瘫痪(PSP)、皮质基底膜变性(CBD)和阿尔茨海默病(AD)与 神经原纤维缠结(NFT)在大脑中的积累。这些集合体主要由以下组成 微管相关蛋白tau(MAPT/tau)，被翻译后修饰严重修饰 (PTMS)。如果我们了解神经元通常是如何调节tau的PTM的，我们或许能够找到恢复 它微妙的平衡。这个项目的目标是了解分子伴侣、热休克 蛋白质70(Hsp70)和热休克蛋白90(Hsp90)直接修饰tau。HSP70和HSP90是已知的 在tau中绑定关键的、易于聚合的位置。虽然这些相互作用提高了tau的溶解度，但它们也 招募一些关键的PTM酶，包括：(I)E3泛素连接酶芯片，(Ii)顺-反式Pro 异构酶FKBP51和(Iii)蛋白磷酸酶PP5。具体来说，这些酶中的每一种都含有一种 四肽重复(TPR)结构域，与C末端保守的EEVD-CO2H基序有亲和力 Hsp70和Hsp90的同源性。因此，我们假设伴侣和TPR结构域之间的复合体最终 决定tau的构象以及它是泛素化还是去磷酸化。得益于 冷冻-EM和最近在大规模多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，以了解伴侣如何与TPR蛋白协调来修饰tau。 在初步研究中，我们测量了伴侣的EEVD基序与一组TPR结构域的结合， 揭示了Hsp70对PP5和Hsp90对FKBP51的意外选择性，以及Hsp70对PP5的关键作用 调节这些亲和力的磷酸化。我们还创建了一个包含约64万个肽序列的文库，并 用它来鉴定芯片的TPR结构域的一个有效的(&lt；10 nM)抑制物，它不能结合密切相关的TPR。 最后，我们验证了一种研究Hsp70-芯片-tau三元复合体的结构方法，这是一个项目 这已经揭示了ADP令人惊讶的、至关重要的作用。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：(SA1)HSP70/HSP90-CHIP，(SA2) HSP90-FKBP51和(SA3)HSP70-PP5。通过这项努力，我们的目标是确定分子开关在 关键的蛋白质接口调节tau的结构和修饰。我们预计这些研究还可以 揭示治疗紧张症的新药物靶点。

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.