Mechanisms of diabetic amyloid formation via 2D IR spectroscopy

通过二维红外光谱研究糖尿病淀粉样蛋白形成机制

• 批准号: 10435538
• 负责人: Martin T Zanni
• 金额: $ 52.58万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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/10435538
• 关键词: Alanine; American; Amyloid; Amyloid Fibrils; Amyloid fibers; Animal Model; Apoptosis; Benign; Beta Cell; Biochemical; Biological Assay; Blood Glucose; Collaborations; Contracts; Cryoelectron Microscopy; Data; Deposition; Diabetes Mellitus; Diagnostic; Disease; Economics; Exhibits; Failure; Genetic Polymorphism; Goals; 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; Membrane; Metabolism; Microscopy; Mind; 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; 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; diabetes mellitus genetics; diabetic; experimental study; glucose tolerance; human tissue; humanized mouse; improved; in vivo; inhibitor; insight; interest; islet; islet amyloid polypeptide; mouse model; mutant; pancreas imaging; prevent; receptor; structural biology; tool

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.

糖尿病淀粉样蛋白形成机制的二维红外光谱研究 摘要 2型糖尿病困扰着近2600万美国人，造成的经济损失比所有癌症都大。 加起来它开始于胰岛素抵抗，但最终胰腺β细胞，使胰岛素失败，导致 显性糖尿病失败部分是由于聚集的激素称为人类胰岛淀粉样蛋白 在一些实施方案中，将多肽（hIAPP或胰淀素）引入淀粉样蛋白斑块中，所述淀粉样蛋白斑块占据高达80%的胰岛空间。令人惊讶的是， 淀粉样纤维本身没有细胞毒性。许多研究人员认为，有毒物质是低聚物， hIAPP，可能通过干扰受体介导的过程或透化膜。因此，在本发明中， 人们对理解hIAPP聚集的机制很感兴趣，因为聚集 途径决定了这些细胞毒性中间体的结构和数量。第一个cryoEM结构 胰淀素是最近报道的，但很少有关于中间体的结构信息，因为应用 大多数结构生物学工具来动态进化蛋白质是困难的。我们发现了一种寡聚物种， 使用我们发明的技术，实时二维红外光谱法，监测hIAPP的聚集动力学。 在这样做的过程中，我们发现hIAPP在FGAIL区域形成具有平行β折叠的寡聚体， 重组成纤维状结构。结构调整的需要导致寿命延长和人口稳定 的低聚物。我们在4种不同的哺乳动物中观察到这种“FGAIL寡聚体”， 糖尿病，加强了我们的假设，即这种中间体是疾病的关键参与者。最重要的是我们 意识到我们可以用一些良性突变来捕获寡聚体。我们被困的低聚物 毒性与野生型hIAPP相同，但在体外持续数天而不是数小时。因为它稳定了这么长时间， 使许多新的结构，生物化学和生理学分析以前不可能。而且，它提供了一个 智力基础，以创建一个新的基因敲入小鼠研究hIAPP寡聚体的动物模型。与 在心中的目标，我们已经开始与人源化小鼠和开发的技术，以图像胰腺 组织与2D IR显微镜。具体目标1将产生一系列捕获的低聚物，其中每一个将被 测试其作为hIAPP寡聚体模型的适用性。具体目标2将研究聚集途径 这导致了最近报道的cryoEM结构，以确定这种多晶型物是否由新的或现有的 机械路径在目标3中，我们通过两个细胞的2D IR成像将我们的体外观察与体内生理学联系起来， 转基因小鼠模型。我们试图从一个基本的角度来理解hIAPP聚合， 对抑制剂设计和激素替代疗法很重要，并利用这些信息来翻译我们的信息。 将体外工作转化为体内动物模型。我们通过机制提供的信息，现在是组织成像， 这是目前任何其他技术都无法实现的。