Single Molecule Studies of IAPP Oligomer Formation and Membrane Permeabilization

描述（由申请人提供）：最近的研究表明，人类胰岛淀粉样多肽（hIAPP）的小可溶性聚集体（低聚物），在胰腺淀粉样蛋白沉积形成之前形成，具有细胞毒性，可能是2型糖尿病病因的关键因素。由于这些hIAPP低聚物的非均质性和瞬态性，用传统技术对其进行详细表征一直具有挑战性。在这个R21中，我们将应用单分子光谱（SMS）方法来深入了解这些有毒物种，沿着以下具体目标：目标1：我们将使用SMS来跟踪溶液中hIAPP低聚物的时间演变，并确定哪些发展成有序结构和/或不溶性淀粉样蛋白沉积。我们将使用荧光标记IAPP的SMS来跟踪低聚物的形成作为时间的函数，并检查与猫(c)和大鼠(r) IAPP相比，NAPP聚集倾向明显增加的基础。需要验证的具体假设是，可溶聚集体通过多种反应途径形成，hIAPP更容易形成初始核（相对于clAPP和rIAPP），从而促进肽聚集成淀粉样蛋白。目的2：研究hIAPP低聚物/原纤维在膜脂质体表面形成的机制，确定导致孔形成和膜渗透的聚集体的大小，以及孔的大小和特异性（如果有的话）。纤维前结构的膜结合被认为是hIAPP细胞毒性的来源。我们将启动SMS实验来验证膜促进hIAPP低聚物形成的假设，从而导致膜表面明确的低聚物结构或原纤维的整合，并且其中一些物种导致膜渗透。目标3。直接观察和表征hIAPP形成的胶束结构，确定其大小分布和临界胶束浓度（CMC）值，并检验这些结构是否可以解释所观察到的h-、r-和clAPP在淀粉样变性方面的差异。我们之前的工作表明，胶束可以缓冲游离单体肽的浓度，从而设置聚集的滞后时间。待验证的假设是，c-和rIAPP的突变导致CMC值降低（从而相应降低单体肽的浓度），从而延缓IAPP的聚集。这也许可以解释为什么这些动物的糖尿病发病率较低。

DESCRIPTION (provided by applicant): Recent studies suggest that small soluble aggregates (oligomers) of human islet amyloid polypeptide (hIAPP), that form before amyloid deposits develop in the pancreas, are cytotoxic and may be critical players in the etiology of Type-2 Diabetes. Due to the heterogeneity and transient nature of these hIAPP oligomers, their detailed characterization by traditional techniques has been challenging. In this R21 we will apply single molecule spectroscopy (SMS) approaches to gain deeper insight into these toxic species along the following specific aims: Aim 1: We will use SMS to follow the time evolution of hIAPP oligomers in solution and to identify which ones develop into ordered structures and/or insoluble amyloid deposit. We will use SMS of fluorescently labeled IAPP to follow the formation of oligomers as a function of time and to examine the basis for the apparent increased propensity for aggregation of NAPP compared to cat (c) and rat(r) IAPP. The specific hypothesis to be tested is that soluble aggregates form by multiple reaction pathways and that hIAPP more readily forms an initial nucleus (relative to clAPP and rIAPP) thus facilitating peptide aggregation into amyloid. Aim 2: To study the mechanism of hIAPP oligomer/protofibril formation on the surface of membrane liposomes and to determine the size of the aggregates leading to pore formation and membrane permeabilization as well as the size and specificity (if any) of the pore. Membrane binding of pre- fibril structures has been suggested as the origin of cytotoxicity of hIAPP. We will initiate SMS experiments to test the hypothesis that membranes facilitate the formation of hIAPP oligomers leading to integration on the membrane surface of well-defined oligomeric structures or protofibrils, and that some of these species lead to membrane permeabilization. Aim 3. To directly observe and characterize the micellar structures formed by hIAPP, to determine their size distribution and critical micelle concentration (CMC) values and to test whether these structures can explain the observed differences in amyloidogenicity between h-, r-, and clAPP. Our previous work revealed that the micelles serve to buffer the concentration of free monomeric peptide and thereby set the lag time for aggregation. The hypothesis to be tested is that the mutations in c- and rIAPP lead to lower CMC values (hence to correspondingly lower concentrations of monomeric peptide) thus retarding IAPP aggregation. This may explain the lower incidence of diabetes in these animals.