Membrane catalyzed amyloid formation in diabetes studied with 2D IR spectroscopy

描述（由申请人提供）：2型糖尿病是美国最流行的疾病之一，造成超过2080万人的痛苦，并在该国的一些地区以流行病的速度扩大。2型糖尿病的关键诊断是胰腺中淀粉样蛋白纤维的存在。这些纤维由人类胰岛淀粉样多肽（hIAPP）组成，许多体外和体内研究已将它们与该疾病联系起来。即便如此，hIAPP抑制胰腺β细胞功能和胰岛素产生的机制尚不清楚。越来越多的证据表明hIAPP与细胞膜的相互作用是导致细胞功能障碍的原因，而不是纤维本身。这一假设的一个证据是脂质囊泡催化纤维的形成，并在此过程中变得可渗透和泄漏。因此，了解疾病机制似乎需要在膜结合、折叠和纤维形成过程中对hIAPP进行结构表征。然而，由于机理是动力学的并且涉及到膜，传统的结构方法如核磁共振很难应用。因此，几乎所有的实验结构信息都来自于圆二色性测量，这只能提供肽结构的基本特征。甚至不清楚肽的哪一部分与膜相结合。考虑到了解hIAPP与脂质膜的结构变化的重要性，我们建议使用FTIR和2D IR光谱，结合1-13C=18O同位素标记，在脂质囊泡存在的折叠动力学过程中获得hIAPP的特定位点结构信息。我们将获得肽与膜的关联、插入和取向、二级结构形成的残基水平信息，并测试膜上是否形成孔隙。结构形成动力学有助于揭示淀粉样蛋白纤维形成的催化机制。我们试图获得hIAPP膜催化动力学的详细结构表征，这是目前其他技术无法实现的。用二维红外光谱研究糖尿病中膜催化淀粉样蛋白的形成。

DESCRIPTION (provided by applicant): Type 2 diabetes is one of the most prevalent diseases in the United States, inflicting more than 20.8 million people and expanding at epidemic rates in some areas of the country. The key diagnostic of Type 2 diabetes is the presence of amyloid fibers in the pancreas. These fibers are composed of the human islet amyloid polypeptide (hIAPP) and many in vitro and in vivo studies have linked them to the disease. Even so, the mechanism by which hIAPP inhibits pancreatic beta-cell function and insulin production is not understood. A growing body of evidence points to hIAPP interacting with the cell membrane as the cause of cell dysfunction rather than the fibers themselves. One piece of evidence for this hypothesis is that lipid vesicles catalyze fiber formation and in doing so become permeable and leak. Thus, it appears that understanding the disease mechanism requires structural characterization of hIAPP during membrane association, folding, and fiber formation. However, since the mechanism is both kinetic and involves membranes, conventional structural approaches such as NMR are difficult to apply. As a result, almost all experimental structural information comes from circular dichroism measurements, which provide only a rudimentary characterization of the peptide structure. It is not even definitively known which part of the peptide associates with the membrane. Considering the importance of understanding the structural changes of hIAPP with lipid membranes, we propose to use FTIR and 2D IR spectroscopy, in conjunction with 1-13C=18O isotope labeling, to yield site-specific structural information on hIAPP during the kinetics of folding in the presence of lipid vesicles. We will gain residue-level information on peptide association with the membrane, insertion and orientation, secondary structure formation, and test whether pores in the membrane form. The kinetics of structure formation will help reveal the catalytic mechanism for amyloid fiber formation. We seek to obtain a detailed structural characterization of hIAPP membrane catalyzed kinetics that is not currently possible with other techniques. Membrane catalyzed amyloid formation in diabetes studied with 2D IR spectroscopy Membrane catalyzed amyloid formation in diabetes studied with 2D IR spectroscopy.