Mechanisms of diabetic amyloid formation studied with 2D IR spectroscopy

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的总和还要大。它始于胰岛素抵抗，但最终导致制造胰岛素的胰岛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多肽(hIAPP或淀粉蛋白)的激素聚集成占据胰岛空间高达80%的淀粉样斑块。令人惊讶的是，淀粉样蛋白纤维本身的细胞毒性低于hIAPP的寡聚体。目前尚不清楚这些寡聚体如何损害β细胞，但它们可能干扰受体介导的过程或渗透代谢膜。因此，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。然而，关于中间体的结构信息很少，因为标准的结构生物学工具很难应用于聚集的蛋白质，更不用说动态进化和膜相关蛋白质了。在上一次资助期间，我们取得了一项技术进步，使我们能够实时收集2D IR光谱，从而监测hIAPP聚集的动力学。我们将我们的光谱与[13]C/[18]O同位素标记相结合，以获得残基特定的结构分辨率。在这样做的过程中，我们有了一个重要的发现：hIAPP的FGAIL区域在进入纤维的无序环之前形成了一个平行的Beta-Sheet中间体。这种无序会在自由能路径上造成很大的势垒，这决定了纤维形成的动力学，并导致中间体的长寿命。我们的数据表明，这种“FGAIL中间体”是目前正在寻找的解释hIAPP毒性的寡聚物种。这也可能是一种理论的关键，该理论解释了为什么一些物种会患2型糖尿病，而另一些物种则不会--这一理论用于设计治疗2型糖尿病的药物。具体目标1将改进这种中间体的结构，并使用体内试验来测试其细胞毒性。特殊目标2将测试来自其他物种的IAPP是否也存在于这种中间体中。最后，特殊目标3利用20红外光谱的能力来研究膜多肽的结构和动力学。当hIAPP聚集在膜小泡上时，我们将绘制具有残基水平特异性的hIAPP的结构图，以确定可能的细胞毒性结构。阐明hIAPP的聚集途径将有助于理解最终导致显性2型糖尿病的β细胞衰竭，并有助于激素替代疗法的开发。我们将获得的结构和动力学将提供目前任何其他技术都不可能实现的hIAPP聚集的详细特征。

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 Beta-cells that make insulin fail, resulting in overt diabetes. Failure is due in part 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 less cytotoxic than are oligomers of hIAPP. It is unknown how these oligomers impair Beta-cells, but they could interfere with receptor mediated processes or permeabolize 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. However, very little structural information exists about intermediates because standard structural biology tools are difficult to apply to aggregated proteins, let alone kinetically evolving and membrane associated proteins. In the last grant period, we made a technological advance that allowed us to collect 2D IR spectra on-the-fly and thereby monitor the kinetics of hIAPP aggregation. We coupled our spectroscopy with [13]C/[18]O isotope labeling to obtain residue specific structural resolution. In doing so, we made an important discovery: the FGAIL region of hIAPP forms a parallel Beta-sheet intermediate before breaking into the disordered loop of the fiber. This disordering causes a large barrier in the free energy pathway which dictates the kinetics of fiber formation and results in a long lifetime for the intermediate. Our data suggests that this "FGAIL intermediate" is the oligomeric species currently being sought to explain hIAPP toxicity. It may also be the key to a theo1y for why some species contract type 2 diabetes but not others - a theory used to design drugs to treat type 2 diabetes. Specific Aim 1 will refine the structure of this intermediate and use in vivo assays to test its cytotoxicity. Specific Aim 2 will test if the IAPP from other species also populates this intermediate. Finally, Specific Aim 3 utilizes the capability of 20 IR spectroscopy for studying membrane peptide structure and kinetics. We will map the structure of hIAPP with residue-level specificity as it aggregates on membrane vesicles to identify possible cytotoxic structures. Elucidating the aggregation pathways of hIAPP will help understand Beta-cell failure that ultimately causes overt type 2 diabetes as well as help in the development of hormone replacement therapies. The structures and kinetics that we will obtain will provide a detailed characterization of hIAPP aggregation that is not currently possible with any other technique.