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，人胰岛淀粉样多肽（hIAPP）的小可溶性聚集体（寡聚体）（在淀粉样蛋白沉积物在胰腺中形成之前形成）具有细胞毒性，可能是2型糖尿病病因学中的关键因素。由于这些hIAPP寡聚体的异质性和瞬时性，通过传统技术对其进行详细表征具有挑战性。在这个R21中，我们将应用单分子光谱（SMS）的方法，以获得更深入的了解这些有毒物质沿着以下具体目标：目标1：我们将使用SMS遵循的时间演变的hIAPP寡聚体在溶液中，并确定哪些发展成有序的结构和/或不溶性淀粉样蛋白存款。我们将使用荧光标记的IAPP的SMS来跟踪作为时间函数的寡聚体的形成，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。待测试的具体假设是可溶性聚集体通过多种反应途径形成，并且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。目标二：研究hIAPP寡聚体/原纤维在膜脂质体表面形成的机制，并确定导致孔形成和膜透化的聚集体的大小以及孔的大小和特异性（如果有的话）。已经提出前原纤维结构的膜结合是hIAPP的细胞毒性的起源。我们将启动SMS实验，以测试的假设，膜促进hIAPP寡聚体的形成，导致整合在明确的寡聚体结构或原纤维的膜表面上，这些物种中的一些导致膜透化。目标3。直接观察和表征hIAPP形成的胶束结构，确定其尺寸分布和临界胶束浓度（CMC）值，并测试这些结构是否可以解释h-、r-和clAPP之间观察到的淀粉样蛋白形成差异。我们以前的工作表明，胶束起到缓冲游离单体肽浓度的作用，从而设定聚集的滞后时间。待测试的假设是c-和rIAPP中的突变导致较低的CMC值（因此相应地降低单体肽的浓度），从而延迟IAPP聚集。这可能解释了这些动物糖尿病发病率较低的原因。

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.