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.

特定蛋白质积累成长纤维状聚集体（称为淀粉样蛋白）是淀粉样蛋白的一个共同特征许多破坏性的与衰老相关的疾病。在 II 型糖尿病中，这些物质的主要成分聚集体是胰岛淀粉样多肽（IAPP，也称为胰淀素）。像许多其他淀粉样蛋白生成一样蛋白质中，IAPP 的聚集与细胞功能障碍和死亡有关。然而，该机制 IAPP 聚集体的形成方式以及这种聚集体与细胞死亡的关系仍然是个谜。帮助为了缩小这一差距，我们建议表征在溶液中形成的人-IAPP 的寡聚中间体，金属（例如锌和铜）的存在，以及通过三个特定目标在脂膜中的存在。 1) 在目标 1 中，我们提议通过核磁共振波谱以原子分辨率表征由 human-IAPP 形成的中间体。将测试鉴定出的寡聚中间体的细胞毒性和衍生自的结构模型 NMR 约束将用于评估淀粉样蛋白抑制剂的机制和效率。 2）由于已经发现锌调节和 II 型糖尿病之间可能存在的遗传联系，我们将描述目的2.通过细胞毒性、NMR等生物物理实验对锌-IAPP加合物进行氧化和氧化的突变体人类 IAPP 的还原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蛋白种类的金属结合亲和力。此外，非原纤维形成且无毒的大鼠-IAPP 和普兰林肽（FDA 批准的商品名 symlin） 1型和2型糖尿病患者）将用作对照。 3）深入了解脂膜辅助 hIAPP 聚集以及 hIAPP 破坏脂膜的机制，我们建议通过各种生物物理技术（包括高速原子技术）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 寡聚中间体并解决通过固态和溶液核磁共振技术的结合，获得低聚物的高分辨率结构。这些高分辨率结构将有助于开发阻止β细胞死亡的药物。