Mechanistic dissection of allosteric modulation and nonproteolytic chaperone activity of human insulin-degrading enzyme

项目摘要。 人胰岛素降解酶（IDE）是一种高度保守的二聚锌金属蛋白酶， 各种肽底物，如胰岛素、淀粉样蛋白-β（Aβ）、胰高血糖素、胰淀素和HIV-1 p6。它牵涉到 几种生理和病理过程，包括促胰岛素释放，淀粉样蛋白-β（Aβ）清除， II型糖尿病和阿尔茨海默病（AD）的发展，以及认知障碍和葡萄糖代谢紊乱。 在艾滋病毒感染者中观察到的不容忍现象。令人惊讶的是，除了水解Aβ外，IDE还充当 一种针对Aβ的非蛋白水解性伴侣，导致其螯合，随后进行受控处置。我们 最近研究了Aβ和HIV-1 p6与无催化活性IDE的相互作用， 溶液NMR方法。我们发现，分子间相互作用的调节使IDE分化为 非淀粉样蛋白p6和淀粉样蛋白Aβ之间的关系。我们还发现， 在亚化学计量浓度下阻止Aβ纤维化。本R21提案中的项目扩展了 这些发现，并将进行创新的IDE的结构-功能研究。具体来说，我们将解决两个 IDE结构生物学领域的突出问题：其催化活性的变构调节（目的 1)以及其对Aβ的显著的非蛋白水解伴侣活性（aim 2）。目标1以我们的假设为中心 底物诱导的一个IDE亚基的关闭将加速另一个IDE亚基的打开， 被释放或基板捕获，并将提供关键的见解如何基板触发这些 构象转变以及亚基内和亚基间相互作用的复杂网络， 控制IDE的催化活性。在目标2下，我们将生成一个详细的量化图片， IDE使用荧光和NMR光谱的协同组合改变Aβ聚集动力学， 基于微观速率定律和化学动力学框架的数学模型。广泛 初步结果，包括生产毫克量的多种催化活性和非活性 IDE变体，以及通过引入稀疏标记的甲基获得的二聚IDE的优异NMR光谱 在全氘环境中，这表明成功完成拟议研究的高度可行性。 鉴于IDE在AD发病机制中的重要性，我们的研究也具有实质性的医学意义 并将为R 01提案奠定必要的基础，该提案旨在弥合生物物理学和生物医学之间的差距。 临床研究和开发用于治疗AD的IDE的选择性激活剂和变体。

Project Summary. Human insulin-degrading enzyme (IDE) is a highly conserved dimeric zinc metalloprotease that hydrolyzes various peptide substrates, such as insulin, amyloid-β (Aβ), glucagon, amylin, and HIV-1 p6. It is implicated in several physiological and pathological processes, including insulin catabolism, amyloid-β (Aβ) clearance, development of type II diabetes and Alzheimer’s disease (AD), as well as cognitive disorders and glucose intolerance observed among people living with HIV. Surprisingly, in addition to hydrolyzing Aβ, IDE also acts as a nonproteolytic chaperone against Aβ, resulting in its sequestration, followed by its controlled disposal. We recently investigated the interactions of Aβ and HIV-1 p6 with catalytically inactive IDE using relaxation-based solution NMR methods. We uncovered that modulation of intermolecular interactions allows IDE to differentiate between non-amyloidogenic p6 and amyloidogenic Aβ. We also discovered that catalytically inactive IDE prevented Aβ fibrillization at substoichiometric concentrations. The projects in this R21 proposal expand upon these discoveries and will carry out innovative structure-function studies of IDE. Specifically, we will address two outstanding questions in the field of IDE structural biology: the allosteric modulation of its catalytic activity (aim 1) and its remarkable nonproteolytic chaperone activity against Aβ (aim 2). Aim 1 is centered on our hypothesis that substrate-induced closure of one IDE subunit will accelerate the opening of the other, allowing the products to be released or substrate captured, and will provide key insights into how the substrate triggers these conformational transitions as well as the complex network of intrasubunit and intersubunit interactions that govern the catalytic activity of IDE. Under aim 2, we will generate a detailed quantitative picture of how inactive IDE alters Aβ aggregation kinetics using a synergistic combination of fluorescence and NMR spectroscopy and mathematical models based on the framework of microscopic rate laws and chemical kinetics. Extensive preliminary results, including the production of milligram quantities of multiple catalytically active and inactive IDE variants, and excellent NMR spectra of dimeric IDE acquired by introducing sparsely labelled methyl groups in a perdeuterated environment, indicate the high feasibility of successfully completing the proposed studies. Given the importance of IDE in the pathogenesis of AD, our research also has substantial medical relevance and will lay the necessary foundation for an R01 proposal, geared toward bridging the gap between biophysical and clinical studies and developing selective activators and variants of IDE for the treatment of AD.