Structural Investigation of Amylin Oligomers Associated to Type-2 Diabetes

与 2 型糖尿病相关的胰淀素寡聚物的结构研究

基本信息

批准号：
10418055
负责人：
Ayyalusamy Ramamoorthy
金额：
$ 38.29万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10418055
关键词：
Affect Affinity Aging Alzheimer&apos s Disease Amyloid Amyloid fibers Atomic Force Microscopy Basic Science Beta Cell Binding Biophysics Calcium Cell Death Cell Nucleus Cell membrane Cells Cellular Membrane Cessation of life Characteristics Chemicals Clinical Research Copper Degenerative Disorder Deposition Diabetes Mellitus Disease Disease Progression FDA approved Fiber Functional disorder Genetic Growth Homeostasis Hormones Human Human Genome Individual Insulin Insulin Resistance Investigation Islet Cell Kinetics Link Lipids Maps Measurement Measures Membrane Membrane Lipids Metal Binding Site Metals Minority Molecular Molecular Chaperones Monitor Multidimensional NMR Techniques Mutation NMR Spectroscopy Names Nature Non-Insulin-Dependent Diabetes Mellitus Nuclear Outcome Oxides Parkinson Disease Pathologic Pathology Pathway interactions Pattern Play Population Pramlintide Process Proteins Rattus Regulation Resolution Risk Factors Role Secretory Vesicles Speed Structural Models Structure Survival Rate Techniques Testing Time Titrations Toxic effect Type 2 diabetic Zinc adduct amyloid formation amyloidogenesis beta pleated sheet biophysical techniques cytotoxic design diabetic diabetic patient drug development experimental study glucose metabolism inhibitor insight insoluble fiber islet amyloid polypeptide misfolded protein mutant nanodisc technology nanodisk non-diabetic protein aggregation solid state solid state nuclear magnetic resonance type I diabetic

项目摘要

The accumulation of particular proteins into long fibrillar aggregates known as amyloids is a common feature of many devastating aging-related pathologies. In type II diabetes mellitus, the main constituent of these aggregates is Islet Amyloid Polypeptide (IAPP, also known as amylin). Like many other amyloidogenic proteins, the aggregation of IAPP has been linked to cellular dysfunction and death. However, the mechanism by which IAPP aggregates form and how this aggregation is linked to cell death remain mysterious. To help reduce this gap, we propose to characterize the oligomeric intermediates of human-IAPP formed in solution, in presence of metals (such as zinc and copper), and in lipid-membrane via three specific aims. 1) In Aim 1, we propose to characterize the intermediates formed by human-IAPP at atomic resolution by NMR spectroscopy. The identified oligomeric intermediates will be tested for cell toxicity and the structural models derived from NMR constraints will be used to evaluate the mechanism and efficiency of amyloid inhibitors. 2) Since a possible genetic link between zinc regulation and type II diabetes has been discovered, we will characterize zinc-IAPP adducts by cell toxicity, NMR and other biophysical experiments in Aim 2. Mutants of oxidized and reduced forms of human-IAPP will be used to probe the metal binding sites, and isothermal titration experiments will be used to measure the metal binding affinities to different amyloid species. In addition, the non-fibril forming and non-toxic rat-IAPP and pramlintide (trade name symlin approved by FDA for use by both type 1 and type 2 diabetic patients) will be used as controls. 3) To gain insight into the lipid-membrane assisted hIAPP aggregation and the mechanism by which hIAPP disrupts the lipid-membrane, we propose to characterize the role of lipid membrane by a variety of biophysical techniques (including high-speed atomic force microscopy), and stabilize hIAPP oligomeric intermediates using lipid-nanodisc technology and solve the high-resolution structure of oligomers by a combination of solid-state and solution NMR techniques. These high-resolution structures will aid in the development of drugs to stop beta-cell death.

特定蛋白质的积累到长纤维骨料中，称为淀粉样蛋白是很常见的特征许多毁灭性的与衰老有关的病理。在II型糖尿病中，其中的主要成分聚集体是胰岛淀粉样多肽（IAPP，也称为淀粉纤维）。像许多其他淀粉样蛋白原蛋白质，IAPP的聚集与细胞功能障碍和死亡有关。但是，机制 IAPP聚集形式以及该聚集与细胞死亡的关系如何仍然是神秘的。提供帮助减少这一差距，我们建议表征在溶液中形成的人类的寡聚中间体金属（例如锌和铜）以及通过三个特定目的在脂质膜中的存在。 1）在AIM 1中，我们提议表征由NMR光谱法原子分辨率在原子分辨率下形成的中间体。确定的低聚物中间体将测试细胞毒性，并从 NMR约束将用于评估淀粉样蛋白抑制剂的机制和效率。 2）自锌调节与II型糖尿病之间可能的遗传联系，我们将表征 AIM 2中的细胞毒性，NMR和其他生物物理实验的锌-IAPP加合物。氧化和减少的人类IAPP的形式将用于探测金属结合位点和等温滴定实验将用于测量与不同淀粉样物种的金属结合亲和力。另外，非纤维形成和无毒的大鼠和pramlintide（由FDA批准的商品名称Symlin供两者使用 1型和2型糖尿病患者）将用作对照。 3）洞悉辅助脂质膜 Hiapp聚集和Hiapp破坏脂质膜的机制，我们建议通过多种生物物理技术（包括高速原子）来表征脂质膜的作用力显微镜），并使用脂质 - 纳米盘技术稳定HIAPP的低聚物中间体并解决通过固态和溶液NMR技术的组合，低分辨率的高分辨率结构。这些高分辨率结构将有助于制定药物制止β细胞死亡。