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RAGE Mediated Mechanisms of Islet Amyloid Polypeptide Cytotoxicity in Type 2 Diab

RAGE介导的2型糖尿病中胰岛淀粉样多肽细胞毒性的机制

基本信息

批准号：
8303204
负责人：
Andisheh Abedini
金额：
$ 5.47万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-23 至 2012-07-22
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8303204
关键词：
Acids Advanced Glycosylation End Products Affect Aliquot Alzheimer&apos s Disease Amyloid Amyloid Fibrils Amyloid deposition Amyloidosis Binding Biochemical Biological Biological Assay Biophysics Brain C2 Domain Cell Death Cell physiology Cell surface Cells Cessation of life Charge Chronic DAG/PE-Binding Domain Data Diabetes Mellitus Disease Dot Immunoblotting Endocrine Epitopes Extracellular Domain Fluorescence Gel Chromatography Goals Graft Survival Health Homeostasis Hormones Human In Vitro Individual Insulin Islets of Langerhans Islets of Langerhans Transplantation Kinetics Learning Ligands Malignant Neoplasms Mediating Methods Molecular Molecular Conformation Nature Neurologic Dysfunctions New York Non-Insulin-Dependent Diabetes Mellitus Outcome Oxidative Stress Pancreas Parkinson Disease Pathogenesis Pathology Peptides Physiological Play Prevention Principal Investigator Process Production Reaction Receptor Activation Recombinants Recruitment Activity Relative (related person)Role Site Solvents Staging Structure System Testing Thioflavin T Time Toxic effect Translating Universities Work amylin receptor amyloid formation analog cytotoxicity exposed human population extracellular follow-up human disease inhibitor/antagonist insight interdisciplinary approach islet islet amyloid polypeptide light scattering monomer mutant neurotoxic novel polypeptide prevent programs receptor receptor binding receptor for advanced glycation endproducts research study small molecule treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Amyloid formation plays an important role in a broad range of human diseases, including Alzheimer's disease (AD), Parkinson's disease, and type 2 diabetes (T2D). Islet amyloid polypeptide (known as amylin or IAPP) is an endocrine hormone responsible for pancreatic amyloid formation in T2D. The process of amyloid formation by human amylin (h-amylin) promotes b-cell death thereby contributing to the loss of 2-cell mass associated with advanced stages of T2D. There is increasing evidence that islet amyloid formation is an important complicating factor in islet cell transplantation, and we have recently shown that prevention of islet amyloid can contribute significantly to graft survival. My goals are to define the nature of the toxic species produced during amyloid formation by h-amylin and to identify the mechanism of islet amyloid induced toxicity in T2D. Biochemical and physiological studies have shown that b-sheet containing amyloid precursors can bind and activate RAGE (receptor for advanced glycation end-products). RAGE is a multi-ligand receptor involved in a diverse range of chronic human disorders including cancers, AD and diabetes. RAGE is a potential cell-surface acceptor site for the neurotoxic amyloid-b (Ab) peptides in the brain of individuals with AD, and plays an important role in the pathogenesis of neurological dysfunction and death. Ab amyloid accumulation in the brain increases with RAGE expression, and activation of RAGE is associated with sustained cellular oxidative stress which adversely affects cellular function and organic homeostasis. Given the similar polypeptide sequences and aggregation kinetics of h-amylin and Ab, I hypothesize that RAGE may also be a pathological receptor for h-amylin; and that activation of RAGE by h-amylin binding is a mechanism of islet amyloid toxicity in T2D. To test this hypothesis, I will apply a multidisciplinary approach which utilizes biological, biophysical and biochemical methods. We have recruited the assistance of a world leader in amylin biophysics to assist us and help guide my studies. My preliminary data show that h-amylin binds RAGE in vitro. I will follow up these results by determining which form of h-amylin (monomer, oligomer and/or amyloid) binds to RAGE and/or has the strongest interaction with RAGE. As part of this aim, I will also identify which extracellular domain(s) of RAGE interact(s) with h-amylin using recombinant soluble RAGE (sRAGE) mutants with one or more domain deletions. Independent of the first aim, I will determine whether h-amylin cytotoxicity is RAGE-mediated. To do this I will first identify the form of h-amylin (monomer, oligomer and/or amyloid) is responsible for cytotoxicity during amyloid formation by using thioflavin-T fluorescence assays, far-UV CD, light scattering and TEM in conjunction with time-dependent assays of cytotoxicity. I will determine the role of RAGE in h-amylin mediated cytotoxicity. These studies will make use of inhibitors of h-amylin toxicity and amyloid formation, as well as mutants of h-amylin which oligomerize but are not toxic. Molecules which trap specific toxic conformations populated by h-amylin during amyloid formation will also be used. The ability of these species to bind the soluble extracellular domain of recombinant human sRAGE will be tested and their effects on cultured RAGE +/+ and RAGE -/- primary cells will be analyzed. This work will provide a molecular level understanding of a receptor-mediated mechanism of islet amyloid toxicity in T2D. The studies will provide a nonbiased strategy for investigating amyloid-RAGE interactions that can be applied to other systems. The lessons learned will not only provide insight into potential strategies for the treatment and prevention of T2D, but may also be translated to better controlling pathological amyloidosis associated with other diseases. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: The goal of this project is to determine whether RAGE plays a role in the pathological amyloid formation and toxicity of human amylin, which is associated with pancreatic islet amyloid and type 2 diabetes. The lessons learned from the proposed studies will provide a nonbiased strategy for investigating amyloid-RAGE interactions that can be applied to other systems. The outcomes of the proposed studies will provide insight into potential strategies for the treatment and prevention of T2D in particular, but may also be translated to better controlling pathological amyloidosis associated with other diseases. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

描述(申请人提供)：淀粉样蛋白的形成在包括阿尔茨海默病(AD)、帕金森病和2型糖尿病(T2D)在内的多种人类疾病中起着重要作用。胰岛淀粉样多肽(又称胰淀素或IAPP)是一种内分泌激素，与T2D胰腺淀粉样蛋白的形成有关。人淀粉样蛋白(h-amylin)形成淀粉样蛋白的过程促进b细胞死亡，从而导致与晚期T2D相关的2细胞质量的损失。越来越多的证据表明，胰岛淀粉样蛋白的形成是胰岛细胞移植中一个重要的复杂因素，我们最近的研究表明，预防胰岛淀粉样蛋白可以显著提高移植物的存活率。我的目标是定义h-淀粉蛋白在淀粉样蛋白形成过程中产生的毒性物种的性质，并确定胰岛淀粉样蛋白在T2D中诱导毒性的机制。生化和生理学研究表明，含有淀粉样前体的b-折叠可以结合和激活RAGE(晚期糖基化终末产物受体)。RAGE是一种多配体受体，与多种慢性人类疾病有关，包括癌症、AD和糖尿病。RAGE是AD患者脑内神经毒性淀粉样蛋白b(Ab)的潜在细胞表面受体，在神经功能障碍和死亡的发病机制中起重要作用。AB淀粉样蛋白在大脑中的积聚随着RAGE表达的增加而增加，RAGE的激活与持续的细胞氧化应激有关，后者对细胞功能和有机稳态产生不利影响。鉴于h-胰淀素和抗体具有相似的多肽序列和聚集动力学，我推测RAGE也可能是h-胰淀素的病理受体；h-淀粉素结合激活RAGE是T2D胰岛淀粉样蛋白毒性的机制之一。为了验证这一假设，我将采用一种利用生物学、生物物理和生化方法的多学科方法。我们已经招募了一位淀粉素生物物理学方面的世界领先者来帮助我们，并帮助指导我的研究。我的初步数据显示，h-胰淀素在体外可以结合RAGE。我将通过确定哪种形式的h-淀粉素(单体、低聚物和/或淀粉样蛋白)与RAGE结合和/或与RAGE具有最强的相互作用来跟进这些结果。作为这一目标的一部分，我还将使用具有一个或多个结构域缺失的重组可溶性RAGE(SRAGE)突变体来鉴定RAGE的哪个胞外结构域(S)与h-淀粉酶(h-Amylin)相互作用(S)。与第一个目标无关，我将确定h-胰淀素的细胞毒性是否由RAGE介导。为此，我将首先使用硫代黄素-T荧光分析、远紫外CD、光散射和电子显微镜结合时间依赖的细胞毒性分析来确定h-淀粉素(单体、低聚物和/或淀粉样蛋白)在淀粉样蛋白形成过程中负责细胞毒性的形式。我将确定RAGE在h-胰淀素介导的细胞毒性中的作用。这些研究将利用h-淀粉素毒性和淀粉样蛋白形成的抑制剂，以及h-淀粉酶的低聚但无毒的突变体。还将使用捕获淀粉样蛋白形成过程中h-淀粉蛋白填充的特定有毒构象的分子。将测试这些物种结合重组人sRAGE的可溶性胞外区的能力，并分析它们对培养的RAGE+/+和RAGE-/-原代细胞的影响。这项工作将从分子水平上理解受体介导的胰岛淀粉样蛋白毒性在T2D中的机制。这些研究将为研究淀粉样蛋白-RAGE相互作用提供一种无偏见的策略，可应用于其他系统。吸取的经验教训不仅将为T2D的治疗和预防提供潜在的策略，而且还可能被转化为更好地控制与其他疾病相关的病理性淀粉样变性。PHS 398/2590(06/09版)页面续格式页面公共卫生相关性：该项目的目标是确定RAGE是否在人淀粉样蛋白的病理性淀粉样蛋白的形成和毒性中发挥作用，这与胰岛淀粉样蛋白和2型糖尿病有关。从拟议的研究中吸取的经验教训将为研究淀粉样蛋白-RAGE相互作用提供一种无偏见的策略，可应用于其他系统。拟议研究的结果将为T2D的治疗和预防提供潜在的策略，但也可能被转化为更好地控制与其他疾病相关的病理性淀粉样变性。PHS 398/2590(06/09版)页面续格式页面