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

描述(申请人提供)：2型糖尿病是美国最流行的疾病之一，造成2,080多万人患病，并在该国一些地区以流行速度扩大。2型糖尿病的关键诊断是胰腺中存在淀粉样纤维。这些纤维是由人类胰岛淀粉样多肽(HIAPP)组成的，许多体外和体内研究已经将它们与疾病联系起来。即便如此，hIAPP抑制胰岛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与细胞膜相互作用是细胞功能障碍的原因，而不是纤维本身。这一假说的一个证据是，脂泡催化纤维的形成，并在此过程中变得可渗透和渗漏。因此，要了解该病的发病机制，似乎需要表征hIAPP在膜结合、折叠和纤维形成过程中的结构特征。然而，由于机理既是动力学的，又涉及膜，常规的结构方法，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圆二色谱测量，这些测量只提供了多肽结构的基本表征。甚至还不能确切地知道多肽的哪一部分与膜有关。考虑到了解hIAPP与脂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，在脂泡存在的情况下，获得hIAPP折叠动力学过程中特定部位的结构信息。我们将获得关于多肽与膜的结合、插入和取向、二级结构形成的残基水平信息，并测试膜中是否有孔形成。结构形成的动力学有助于揭示淀粉样纤维形成的催化机理。我们试图获得hIAPP膜催化动力学的详细结构特征，这是目前其他技术无法实现的。膜催化糖尿病淀粉样蛋白形成的二维红外光谱研究膜催化的糖尿病淀粉样蛋白的形成。

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.