Single Molecule Studies of IAPP Oligomer Formation and Membrane Permeabilization

IAPP 寡聚物形成和膜透化的单分子研究

• 批准号: 7074118
• 负责人: ARI GAFNI
• 金额: $ 22.13万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7074118
• 关键词: amylin; charge coupled device camera; colloids; cytotoxicity; disease /disorder etiology; fluorescence; fluorescence resonance energy transfer; gene mutation; intermolecular interaction; liposomes; membrane permeability; membrane proteins; noninsulin dependent diabetes mellitus; pancreatic polypeptide; peptide chemical synthesis; pore forming protein; protein structure; spectrometry; tissue /cell preparation

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.

描述(申请人提供)：最近的研究表明，在胰腺中形成淀粉样沉积之前形成的人胰岛淀粉样多肽(HIAPP)的可溶小聚体(寡聚体)具有细胞毒性，可能是2型糖尿病的病因学关键因素。由于这些hIAPP寡聚体的异质性和瞬变性质，用传统技术对其进行详细的表征是具有挑战性的。在本R21中，我们将应用单分子光谱(SMS)方法，沿着以下特定目标更深入地了解这些有毒物种：目标1：我们将使用单分子光谱跟踪溶液中hIAPP低聚物的时间演变，并确定哪些低聚物发展为有序结构和/或不溶淀粉样蛋白沉积。我们将使用荧光标记的IAPP的SMS来跟踪低聚物的形成作为时间的函数，并检查与CAT(C)和RAT(R)IAPP相比，NAPP的聚集倾向明显增加的基础。需要检验的具体假设是，可溶性聚集体由多条反应途径形成，hIAPP更容易形成初始核(相对于Clapp和rIAPP)，从而促进多肽聚集成淀粉样蛋白。目的：研究hIAPP齐聚物/原纤维在膜脂质体表面的形成机制，确定导致膜通透性和孔道形成的聚集体的大小以及孔道的大小和特异性(如果有的话)。前丝原结构的膜结合被认为是hIAPP细胞毒性的来源。我们将启动SMS实验来测试这一假设，即膜促进hIAPP寡聚体的形成，导致整合到定义明确的寡聚体结构或原纤维的膜表面，并且其中一些物种导致膜通透性。目的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h-、r-和Clapp之间淀粉样变性的差异。我们先前的工作表明，胶束可以缓冲游离单体多肽的浓度，从而设定聚集的滞后时间。需要检验的假设是，c-和rIAPP的突变导致CMC值降低(从而相应地降低了单体多肽的浓度)，从而延缓了IAPP的聚集。这可能解释了为什么这些动物的糖尿病发病率较低。