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，在胰腺中形成淀粉样蛋白沉积物之前形成的人胰岛淀粉样蛋白多肽（hIAPP）的小可溶性聚集体（寡聚物）具有细胞毒性，并且可能是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一直具有挑战性。在此 R21 中，我们将应用单分子光谱 (SMS) 方法，按照以下具体目标更深入地了解这些有毒物质： 目标 1：我们将使用 SMS 跟踪溶液中 hIAPP 低聚物的时间演化，并识别哪些低聚物发展成有序结构和/或不溶性淀粉样蛋白沉积物。我们将使用荧光标记 IAPP 的 SMS 来跟踪寡聚体的形成随时间的变化，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。要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 2：研究 hIAPP 寡聚物/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）。前原纤维结构的膜结合被认为是 hIAPP 细胞毒性的根源。我们将启动 SMS 实验来测试以下假设：膜促进 hIAPP 寡聚物的形成，从而导致明确的寡聚结构或原纤维整合在膜表面，并且其中一些物种会导致膜透化。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观察到的 h-、r- 和 clAPP 之间淀粉样蛋白形成性的差异。我们之前的工作表明，胶束可以缓冲游离单体肽的浓度，从而设定聚集的滞后时间。要测试的假设是 c- 和 rIAPP 中的突变导致 CMC 值降低（因此单体肽浓度相应降低），从而阻碍 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.