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Mechanisms of diabetic amyloid formation studied with 2D IR spectroscopy

用二维红外光谱研究糖尿病淀粉样蛋白形成机制

基本信息

批准号：
10862345
负责人：
Martin T Zanni
金额：
$ 2.5万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-03-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10862345
关键词：
Alanine American Amyloid Amyloid Fibrils Amyloid fibers Animal Model Apoptosis Benign Beta Cell Biochemical Biological Assay Blood Glucose Cell Membrane Permeability Collaborations Contracts Cryoelectron Microscopy Data Deposition Diabetes Mellitus Diagnostic Disease Economics Excretory function Exhibits Failure Genetic Genetic Polymorphism Grant Hand Homeostasis Hormone replacement therapy Hormones Hour Human Image Impairment In Vitro Individual Insulin Insulin Resistance Investigation Islets of Langerhans Transplantation Isotope Labeling Kinetics Knock-in Mouse Knowledge Laboratories Link Location Malignant Neoplasms Mediating Metabolism Microscopy Modeling Monitor Mus Mutation NMR Spectroscopy Non-Insulin-Dependent Diabetes Mellitus Pancreas Pathway interactions Physiological Physiology Play Polymorph Population Process Production Proteins Publishing Reporting Research Research Personnel Role Senile Plaques Series Sorting Spectrum Analysis Stable Populations Structure Structure of beta Cell of islet Techniques Technology Testing Thinking Tissue imaging Tissues Toxic effect Transgenic Mice Translating Work aggregation pathway amyloid formation beta pleated sheet combat cytotoxic cytotoxicity design diabetic experimental study glucose tolerance human tissue humanized mouse improved in vivo inhibitor insight interest invention islet islet amyloid polypeptide mouse model mutant pancreas imaging prevent receptor structural biology tool

项目摘要

Mechanisms of diabetic amyloid formation via 2D IR spectroscopy Abstract Type 2 diabetes afflicts nearly 26 million Americans and causes a larger economic loss than all cancers combined. It starts as insulin resistance, but ultimately the pancreatic β-cells that make insulin fail, resulting in overt diabetes. Failure is partially due to aggregation of the hormone known as the human islet amyloid polypeptide (hIAPP or amylin) into amyloid plaques that occupy up to 80% of the islet space. Surprisingly, the amyloid fibers themselves are not cytotoxic. Many researchers believe that the toxic species are oligomers of hIAPP, perhaps by interfering with receptor mediated processes or permeabilizing the membrane. As a result, there is much interest in understanding the mechanism by which hIAPP aggregates, because the aggregation pathway dictates the structures and populations of these cytotoxic intermediates. The first cryoEM structure of amylin was recently reported, but very little structural information exists about intermediates because applying most structural biology tools to kinetically evolving proteins is difficult. We discovered an oligomeric species by monitoring the aggregation kinetics of hIAPP using a technology that we invented, on-the-fly 2D IR spectroscopy. In doing so, we discovered that hIAPP forms oligomers with a parallel β-sheet in the FGAIL region, prior to restructuring into its fibrillar structure. The need to restructure results in a prolonged lifetime and stable population of the oligomers. We observed this “FGAIL oligomer” in 4 different mammalian species known to contract type 2 diabetes, strengthening our hypothesis that this intermediate is a key player in the disease. Most importantly, we realized that we could trap the oligomer with a few benignly placed mutations. Our trapped oligomers are nearly as toxic as wild-type hIAPP, but persist in vitro for days rather than hours. Because it is stable for so long, it enables many new structural, biochemical, and physiological assays not previously possible. And, it provides an intellectual basis to create a new knock-in mouse to investigate hIAPP oligomers in an animal model. With that goal in mind, we have begun working with humanized mice and developed the technology to image pancreas tissues with 2D IR microscopy. Specific Aim 1 will generate a series of trapped oligomers, each of which will be tested for its suitability as a model for hIAPP oligomers. Specific Aim 2 will investigate the aggregation pathway that leads to a recently reported cryoEM structure to determine if this polymorph is formed from a new or existing mechanistic pathway. In Aim 3, we link our in vitro observations to in vivo physiology via 2D IR imaging of two transgenic mouse models. We seek to understand hIAPP aggregation from a fundamental perspective, which is important for inhibitor design and hormone replacement therapies, and utilize that information to translate our in vitro work into in vivo animal models. The information that we provide via mechanisms, and now tissue imaging, is currently not possible with any other technique.

通过二维红外光谱研究糖尿病淀粉样蛋白形成机制抽象的 2 型糖尿病困扰着近 2600 万美国人，其造成的经济损失比所有癌症都大合并。它一开始是胰岛素抵抗，但最终导致胰岛素失效的胰腺 β 细胞，导致明显的糖尿病。失败的部分原因是人类胰岛淀粉样蛋白的聚集多肽（hIAPP 或胰岛淀粉样多肽）转化为淀粉样斑块，占据高达 80% 的胰岛空间。令人惊讶的是，淀粉样纤维本身不具有细胞毒性。许多研究人员认为，有毒物质是以下物质的低聚物： hIAPP，可能是通过干扰受体介导的过程或透化膜来实现的。因此，人们对理解 hIAPP 聚合的机制很感兴趣，因为聚合途径决定了这些细胞毒性中间体的结构和数量。第一个冷冻电镜结构最近报道了胰淀素，但有关中间体的结构信息很少，因为应用大多数动态进化蛋白质的结构生物学工具都很困难。我们发现了一种寡聚物种使用我们发明的动态 2D 红外光谱技术监测 hIAPP 的聚集动力学。在此过程中，我们发现 hIAPP 在 FGAIL 区域形成具有平行 β-折叠的寡聚物，先于重组为其纤维结构。重组的需要导致寿命延长和人口稳定的低聚物。我们在 4 种已知患有 2 型感染的不同哺乳动物物种中观察到这种“FGAIL 寡聚体” 糖尿病，加强了我们的假设，即这种中间体是该疾病的关键参与者。最重要的是，我们意识到我们可以通过一些良性突变来捕获寡聚物。我们捕获的低聚物几乎与野生型 hIAPP 具有相同的毒性，但在体外可以持续数天而不是数小时。因为长期稳定，所以使许多以前不可能的新结构、生化和生理测定成为可能。并且，它提供了一个创造新的敲入小鼠以在动物模型中研究 hIAPP 寡聚物的智力基础。有了那个牢记目标，我们已经开始与人源化小鼠合作并开发胰腺成像技术使用 2D IR 显微镜观察组织。具体目标 1 将生成一系列捕获的低聚物，其中每一个都将测试了其作为 hIAPP 寡聚物模型的适用性。具体目标 2 将研究聚集途径这导致了最近报道的冷冻电镜结构，以确定这种多晶型物是否是由新的或现有的形成的机械途径。在目标 3 中，我们通过两个细胞的 2D IR 成像将体外观察与体内生理学联系起来。转基因小鼠模型。我们试图从基本角度理解 hIAPP 聚合，即对于抑制剂设计和激素替代疗法很重要，并利用该信息来转化我们的体外工作进入体内动物模型。我们通过机制提供的信息，现在是组织成像，目前任何其他技术都无法实现。