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Probing the role of helix-1 in amyloid formation by serum amyloid A

通过血清淀粉样蛋白 A 探讨 helix-1 在淀粉样蛋白形成中的作用

基本信息

批准号：
9098058
负责人：
RUEL Z B DESAMERO
金额：
$ 49.32万
依托单位：
YORK COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9098058
关键词：
Acute-Phase Proteins Americas Amino Acids Amyloid Amyloid Fibrils Amyloidosis Arginine Binding Biochemical Biological Assay Chemicals Chronic Collaborations Complication Computer Analysis Computer Simulation Critical Thinking Data Deposition Development Disease Employment Failure Fluorescence Resonance Energy Transfer Foundations Goals Inflammatory Journals Kinetics Knowledge Label Laboratories Laboratory Chemicals Lead Learning Life Methods Modeling Morphology N-terminal Organ Peptide Synthesis Peptides Phase Play Point Mutation Process Property Proteins Research Research Activity Research Infrastructure Research Personnel Role Science Series Serum amyloid A protein Solid Spectrum Analysis Structure Students Techniques Technology Theoretical model Therapeutic Agents Tissues Transmission Electron Microscopy United States United States National Institutes of Health Work alpha helix amyloid formation base community college experience high school improved inhibitor/antagonist molecular dynamics monomer mutant peptide analog polypeptide programs public health relevance self assembly spectroscopic survey symposium synthetic peptide theories underrepresented minority student university student

项目摘要

DESCRIPTION (provided by applicant): AA amyloidosis is a severe complication of chronic inflammatory disorders and is potentially fatal. The amyloid fibrils involved in AA amyloidosis are derived from serum amyloid A (SAA) which is an acute phase reactant protein. In AA amyloidosis, circulating amyloid fibrils are deposited in organs ultimately leading to their failur. Serum amyloid A (SAA) is the major protein component of amyloid fibrils associated with AA amyloidosis. Recent molecular dynamics simulations have provided an in silico model for SAA misfolding in which the N-terminal helical region (Helix-1) of the molecule plays a pivotal role. This portion of the polypeptide is believed to transition from a α-helix to β-hairpin. The β-haipin units are postulated associate and stack upon themselves to produce amyloid fibrils. It is the focus of this proposal to experimentally investigate this model of SAA misfolding through the synthesis and characterization of peptide analogs of the helix-1 region. The roles of specific amino acids will be probed and their trajectory during SAA misfolding interrogated. Peptides containing specific point mutations along with isotopically labeled residues will be employed in conjunction with thioflavin T kinetic aggregation assays, FRET, Raman and IR vibrational spectroscopic studies to determine the key molecular interactions and mechanism responsible for SAA fibrillization. Transmission electron microscopy will also be employed to determine the morphology of amyloid fibrils. Experimental data will be compared and contrasted to the in silico model. One of the major goals of this project is to provide underrepresented minority students, >50% of our student body, with the opportunity to conduct research and gain experience working in a modern chemical laboratory. Students involved in this project will be exposed to a variety of techniques ranging from solid phase peptide synthesis and kinetic assays to advanced spectroscopic and biochemical methods. This project provides students the opportunity to apply theories learned in the class room to real world chemical problems in the laboratory. Hands-on research experience is one of the most effective approaches for the development of critical thinking and improving the scientific infrastructure of the United States o America. Moreover, research experiences that are based on current technology allow students to solidify and build on their knowledge in ways that enhances a connection to the real world. This experience will help prepare students for graduate programs and employment in the chemical sciences. The educational impact of this project also extends to local high school and community college students already working in the PIs laboratories. These high school and community college students benefit from their direct collaboration with NIH-AREA trainees and by attending all scholarly activities of research groups, such as seminars, conferences and journal clubs.

描述（由申请人提供）：AA 淀粉样变性是慢性炎症性疾病的严重并发症，可能致命。参与 AA 型淀粉样变性的淀粉样原纤维是源自血清淀粉样蛋白 A (SAA)，它是一种急性期反应蛋白。在 AA 型淀粉样变性中，循环淀粉样原纤维沉积在器官中，最终导致器官衰竭。血清淀粉样蛋白 A (SAA) 是与 AA 淀粉样变性相关的淀粉样原纤维的主要蛋白质成分。最近的分子动力学模拟提供了 SAA 错误折叠的计算机模型，其中分子的 N 端螺旋区域 (Helix-1) 起着关键作用。据信多肽的这一部分从α-螺旋转变为β-发夹。假设 β-haipin 单元相互关联并堆叠以产生淀粉样原纤维。本提案的重点是通过 helix-1 区域肽类似物的合成和表征来实验研究 SAA 错误折叠模型。将探究特定氨基酸的作用并探究它们在 SAA 错误折叠过程中的轨迹。含有特定点突变和同位素标记残基的肽将与硫黄素 T 动力学聚集测定、FRET、拉曼和红外振动光谱研究结合使用，以确定导致 SAA 纤维化的关键分子相互作用和机制。透射电子显微镜也将用于确定淀粉样原纤维的形态。实验数据将与计算机模型进行比较和对比。该项目的主要目标之一是为占学生总数 50% 以上的少数族裔学生提供进行研究并获得在现代化学实验室工作经验的机会。参与该项目的学生将接触到各种技术，从固相肽合成和动力学测定到先进的光谱和生化方法。该项目为学生提供了将课堂上学到的理论应用于实验室中现实世界化学问题的机会。实践研究经验是发展批判性思维和改善美国科学基础设施的最有效方法之一。此外，基于当前技术的研究经验使学生能够巩固和发展他们的知识，从而增强与现实世界的联系。这种经验将帮助学生为化学科学领域的研究生课程和就业做好准备。该项目的教育影响还延伸到了已经在 PI 实验室工作的当地高中和社区学院的学生。这些高中和社区学院的学生受益于与 NIH-AREA 学员的直接合作以及参加研究小组的所有学术活动，例如研讨会、会议和期刊俱乐部。