Mechanisms of diabetic amyloid formation via 2D IR spectroscopy

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要大 加在一起。它始于胰岛素抵抗，但最终导致制造胰岛素的胰腺β细胞衰竭，导致 明显的糖尿病。失败的部分原因是被称为人类胰岛淀粉样蛋白的激素聚集。 多肽(hIAPP或胰淀素)转化为淀粉样斑块，占据胰岛空间的80%。令人惊讶的是， 淀粉样纤维本身并不具有细胞毒性。许多研究人员认为，这些有毒物种是低聚的 HIAPP，可能通过干扰受体介导的过程或使膜通透。结果, 人们对了解hIAPP聚集的机制很感兴趣，因为聚集 途径决定了这些细胞毒中间体的结构和种群。的第一个低温电子显微镜结构 胰淀素是最近报道的，但关于中间体的结构信息很少，因为应用 大多数结构生物学工具都很难用来动态进化蛋白质。我们发现了一种寡聚物种，通过 使用我们发明的动态二维红外光谱技术监测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-Sheet的低聚物，在此之前 重组成纤维状结构。重组的需要导致寿命延长和人口稳定 低聚物的含量。我们在4种已知感染2型病毒的哺乳动物中观察到了这种“FGAIL寡聚体”。 糖尿病，强化了我们的假设，即这种中间体是疾病的关键参与者。最重要的是，我们 意识到我们可以通过一些有利的位置突变来捕获寡聚体。我们捕获的低聚物几乎 毒性与野生型hIAPP相同，但在体外可持续数天而不是数小时。因为它稳定了这么长时间，所以它 使许多以前不可能实现的新的结构、生化和生理分析成为可能。而且，它还提供了一个 创造一种新的敲入小鼠的智力基础，以在动物模型中研究hIAPP寡聚体。有了这些， 考虑到目标，我们已经开始对人源化的小鼠进行研究，并开发了成像胰腺的技术 组织进行二维红外显微镜观察。特定目标1将产生一系列捕获的寡聚体，每个寡聚体都将 测试了其作为hIAPP齐聚物模型的适用性。《特定目标2》将研究聚集途径 这导致了最近报道的一种低温EM结构，以确定这种多晶型是由新的还是现有的 机械路径。在目标3中，我们通过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。我们试图从根本的角度理解hIAPP聚合，这是 对于抑制剂设计和激素替代疗法很重要，并利用这些信息将我们的 体外工作变成活体动物模型。我们通过机制提供的信息，现在是组织成像， 目前用任何其他技术都不可能。

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.