Mechanisms of diabetic amyloid formation studied with 2D IR spectroscopy

用二维红外光谱研究糖尿病淀粉样蛋白形成机制

• 批准号: 9031099
• 负责人: Martin T Zanni
• 金额: $ 36.98万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9031099
• 关键词: Address; American; Amino Acids; Amyloid; Amyloid fibers; Amyloidosis; Animals; Apoptosis; Beta Cell; Binding; Biological Assay; Canis familiaris; Cell Death; Cell Nucleus; Cells; Collaborations; Contracts; Coupled; Data; Deposition; Development; Diabetes Mellitus; Diagnostic; Disease; Drug Design; Economics; Energy Metabolism; FDA approved; Failure; Felis catus; Fiber; Free Energy; Grant; Homeostasis; Hormone replacement therapy; Hormones; Human; In Vitro; Individual; Insulin; Isotope Labeling; Kinetics; Knowledge; Lipids; Malignant Neoplasms; Mammals; Maps; Mediating; Membrane; Membrane Proteins; Monitor; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Papio; Pathway interactions; Peptides; Pharmaceutical Preparations; Play; Population; Process; Production; Research; Resolution; Role; Senile Plaques; Specificity; Spectrum Analysis; Structure; Techniques; Testing; Toxic effect; Transplantation; Variant; Vesicle; aggregation pathway; alpha helix; amyloid formation; beta pleated sheet; combat; cytotoxic; cytotoxicity; cytotoxicity test; diabetic; improved; in vivo; inhibitor/antagonist; insight; interest; islet; islet amyloid polypeptide; peptide structure; prevent; protein aggregate; receptor; research study; structural biology; theories; tool

 DESCRIPTION (provided by applicant): Type 2 diabetes afflicts nearly 26 million Americans and causes a larger economic loss than all cancers combined. It starts as insulin but ultimately the pancreatic -cells that make insulin fail, resulting in overt diabetes. Failure is due in par to aggregation of the hormone known as the human islet amyloid polypeptide (hIAPP or amylin) into amyloid plaques that occupy up to 80% of the islet space. Surprisingly, the amyloid fibers themselves are less cytotoxic than are oligomers of hIAPP. It is unknown how these oligomers impair -cells, but they could interfere with receptor mediated processes or permeabolize the membrane, As a result, there is much interest in understanding the mechanism by which hIAPP aggregates, because the aggregation pathway dictates the structures and populations of these cytotoxic intermediates. However, very little structural information exists about intermediates because standard structural biology tools are difficult to apply to aggregated proteins, let alone kinetically evolving and membrane associated proteins. In the last grant period, we made a technological advance that allowed us to collect 2D IR spectra on-the-fly and thereby monitor the kinetics of hIAPP aggregation. We coupled our spectroscopy with 13C/18O isotope labeling to obtain residue specific structural resolution. In doing so, we made an important discovery the FGAIL region of hIPP forms a parallel -sheet intermediate before breaking into the disordered loop of the fiber. This disordering causes a large barrier in the free energy pathway which dictates the kinetics of fiber formation and results in a long lifetime for the intermediate. Our dta suggest that this "FGAIL intermediate" is the oligomeric species currently being sought to explain hIAPP toxicity. It may also be the key to a theory for why some species contract type 2 diabetes but not others - a theory used to design drugs to treat type 2 diabetes. Specific Aim 1 will refine the structure of this intermediate and use in vivo assays to test its cytotoxicity. Specific Aim 2 will test if the IAPP from other species also populates this intermediate. Finally, Specific Aim 3 utilizes the capability of 2D IR spectroscopy for studying membrane peptide structure and kinetics. We will map the structure of hIAPP with residue-level specificity as it aggregates on membrane vesicles to identify possible cytotoxic structures. Elucidating the aggregation pathways of hIAPP will help understand -cell failure that ultimately causes overt type 2 diabetes as well as help in the development of hormone replacement therapies. The structures and kinetics that we will obtain will provide a detailed characterization of hIAPP aggregation that is not currently possible with any other technique.

 2型糖尿病困扰着近2600万美国人，造成的经济损失比所有癌症的总和还要大。它开始于胰岛素，但最终胰腺β细胞，使胰岛素失败，导致明显的糖尿病。失败是由于被称为人胰岛淀粉样多肽（hIAPP或胰淀素）的激素聚集成淀粉样斑块，其占据高达80%的胰岛空间。令人惊讶的是，淀粉样蛋白纤维本身的细胞毒性比hIAPP的寡聚体小。这些寡聚体如何损害β-细胞尚不清楚，但它们可能干扰受体介导的过程或使膜透化。因此，人们对理解hIAPP聚集的机制非常感兴趣，因为聚集途径决定了这些细胞毒性中间体的结构和群体。然而，关于中间体的结构信息很少，因为标准结构生物学工具很难应用于聚集的蛋白质，更不用说动力学进化和膜相关蛋白质了。在上一个资助期，我们取得了技术进步，使我们能够收集二维红外光谱的飞行，从而监测hIAPP聚集的动力学。我们将光谱学与13 C/18 O同位素标记结合起来，以获得残基特定的结构分辨率。在这样做的过程中，我们有了一个重要的发现，hIPP的FGAIL区域在断裂成纤维的无序环之前形成平行的折叠中间体。这种无序导致自由能途径中的大势垒，其决定纤维形成的动力学并导致中间体的长寿命。我们的数据表明，这种“FGAIL中间体”是目前正在寻求解释hIAPP毒性的寡聚物种类。这也可能是为什么一些物种患2型糖尿病而其他物种不患2型糖尿病的理论的关键--这一理论用于设计治疗2型糖尿病的药物。具体目标1将细化该中间体的结构，并使用体内试验来测试其细胞毒性。 特定目标2将检测来自其他种属的IAPP是否也填充该中间体。最后，具体目标3利用二维红外光谱研究膜肽结构和动力学的能力。我们将映射的结构与残留水平的特异性，因为它聚集在膜囊泡，以确定可能的细胞毒性结构的hIAPP。阐明hIAPP的聚集途径将有助于理解最终导致明显2型糖尿病的胰岛细胞衰竭，并有助于激素替代疗法的发展。我们将获得的结构和动力学将提供hIAPP聚集的详细表征，这是目前任何其他技术都不可能实现的。