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

人胰岛素降解酶变构调节和非蛋白水解伴侣活性的机制剖析

• 批准号: 10667987
• 负责人: Lalit Deshmukh
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667987
• 关键词: Abeta clearance; Abeta synthesis; Acceleration; Age; Aging; Allosteric Regulation; Alzheimer' s Disease; Alzheimer' s disease pathology; Amyloid; Amyloid beta-Protein; Antibodies; Brain; Catabolism; Cerebrum; Characteristics; Chemicals; Clinical Research; Cognition Disorders; Complex; Cryoelectron Microscopy; Deterioration; Development; Dissection; Endopeptidases; Environment; Enzyme Inhibition; Enzymes; Exhibits; Exocytosis; Fluorescence Spectroscopy; Foundations; Functional disorder; Genetic Polymorphism; Glucagon; Glucose Intolerance; HIV; HIV-1; Heat shock proteins; Human; Human Activities; In Vitro; Insulin; Insulinase; Kinetics; Knockout Mice; Label; Late Onset Alzheimer Disease; Laws; Ligands; Mediating; Medical; Metabolic Diseases; Metalloproteases; Methods; Microscopic; Molecular; Molecular Chaperones; Molecular Conformation; NMR Spectroscopy; Neurodegenerative Disorders; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Pathogenesis; Pathologic Processes; Pathology; Pathway interactions; Peptides; Persons; Physiological Processes; Play; Polyphosphates; Prevention; Production; Property; Proteins; Proteome; Relaxation; Research; Risk; Roentgen Rays; Role; Senile Plaques; Site; Structure; System; Therapeutic; Variant; Zinc; abeta accumulation; abeta deposition; abeta oligomer; beta-Endorphin; biophysical analysis; biophysical techniques; chemical kinetics; conformational conversion; design; dimer; enzyme activity; enzyme structure; innovation; insight; insulin dimers; intermolecular interaction; islet amyloid polypeptide; mathematical model; methyl group; milligram; monomer; novel therapeutic intervention; overexpression; prevent; proteostasis; structural biology

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.

项目摘要。 人胰岛素降解酶 (IDE) 是一种高度保守的二聚体锌金属蛋白酶，可水解 多种肽底物，如胰岛素、β-淀粉样蛋白 (Aβ)、胰高血糖素、胰淀素和 HIV-1 p6 都有涉及。 多种生理和病理过程，包括胰岛素分解代谢、β-淀粉样蛋白 (Aβ) 清除、 II 型糖尿病和阿尔茨海默病 (AD) 以及认知障碍和血糖的发展 令人惊讶的是，在 HIV 感染者中观察到的不耐受现象，除了水解 Aβ 之外，IDE 还具有以下作用： 针对 Aβ 的非蛋白水解伴侣，导致其隔离，然后进行受控处置。 最近使用基于松弛的方法研究了 Aβ 和 HIV-1 p6 与催化失活 IDE 的相互作用 我们发现分子间相互作用的调节使 IDE 能够区分。 我们还发现了催化失活的 IDE。 在亚化学计量浓度下防止 Aβ 纤维化 该 R21 提案中的项目扩展了该项目。 这些发现将针对 IDE 进行创新的结构功能研究，具体而言，我们将解决两个问题。 IDE结构生物学领域的突出问题：其催化活性的变构调节（目标 1) 及其针对 Aβ 的显着非蛋白水解伴侣活性（目标 1）以我们的假设为中心。 底物诱导的一个 IDE 子单元的关闭将加速另一个 IDE 子单元的打开，从而使产品 被释放或底物被捕获，并将提供关于底物如何触发这些的关键见解 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，我们将生成关于不活跃程度的详细定量图。 IDE 利用荧光和 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广泛化学动力学框架的数学模型。 初步结果，包括毫克量的多种催化活性和非活性物质的生产 IDE 变体，以及通过引入稀疏标记的甲基获得的二聚 IDE 的出色 NMR 谱 在置换环境中，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，我们的研究也具有重大的医学意义 并将为 R01 提案奠定必要的基础，旨在缩小生物物理之间的差距 临床研究和开发用于治疗 AD 的选择性激活剂和 IDE 变体。