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

• 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
• 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' 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

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.

特定蛋白质积累成称为淀粉样蛋白的长纤维状聚集体是淀粉样蛋白的共同特征。 许多与衰老有关的毁灭性疾病在II型糖尿病中， 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 由于IAPP是一种蛋白质，IAPP的聚集与细胞功能障碍和死亡有关。然而，机制 IAPP聚集体是如何形成的，以及这种聚集如何与细胞死亡联系在一起仍然是个谜。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， 存在的金属（如锌和铜），并通过三个特定的目的在脂质膜。1)目标1： 建议通过NMR光谱以原子分辨率表征由人-IAPP形成的中间体。 将测试鉴定的低聚中间体的细胞毒性和衍生自以下的结构模型： 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 锌调节和II型糖尿病之间可能存在遗传联系，我们将描述 锌-IAPP加合物的细胞毒性，NMR和其他生物物理实验目的2。氧化和 还原形式的人-IAPP将用于探测金属结合位点， 实验将用于测量金属与不同淀粉样物质的结合亲和力。此外该 非纤维形成和无毒的大鼠-IAPP和普兰林肽（FDA批准的商品名symlin， 1型和2型糖尿病患者）用作对照。3)为了深入了解脂质膜辅助的 hIAPP聚集和hIAPP破坏脂质膜的机制，我们建议 通过各种生物物理技术（包括高速原子吸收光谱技术）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寡聚体中间体，并解决 通过固态和溶液NMR技术的组合来高分辨率地构造低聚物。这些 高分辨率结构将有助于开发阻止β细胞死亡的药物。