AFM AND MS TO MONITOR FIBRIL FORMATION FROM AMYLOID IG LIGHT CHAINS AND GAGS

AFM 和 MS 监测淀粉样蛋白 IG 轻链和 GaGS 的纤维形成

• 批准号: 7955940
• 负责人: Vickery E Trinkaus-Randall
• 金额: $ 1.13万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955940
• 关键词: Aliquot; Amyloid; Amyloid Fibrils; Amyloid Proteins; Biology; Buffers; Caliber; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Data; Filament; Funding; Gagging; Glycosaminoglycans; Grant; Heparin; Human; Immunoglobulin G; In Vitro; Inorganic Sulfates; Institution; Light; Mass Spectrum Analysis; Measures; Medicine; Methods; Modeling; Molecular Conformation; Monitor; Organ; Patients; Process; Proteins; Recombinants; Reporting; Research; Research Personnel; Resources; Solutions; Source; Structure; Testing; Time; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; amyloid fibril formation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Amyloid fibrils are composed of abnormally refolded proteins. AFM has demonstrated its capability to elucidate the in vitro fibril formation process. The fibril formation of amyloid protein such as Amyloid-¿ and recombinant IgG lightchain has been reported. The model proposed by Ionescu-Zanetti et al shows that the amyloid proteins form ¿-sheet structures to become a single filament in the diameter around 2.4 nm. Two or more filaments can intertwine to form larger size protofilbrils or fibrils directly. This AFM project is related to our other amyloid mass spectrometry projects, and focuses on IgG light-chains purified from patient organs and resuspended in solution, as well as the fibrils taken directly from human organs. AL LC fibrils are suspended in buffer and aliquots are taken at different times for AFM analysis under tapping mode. Our preliminary data showed that the rate of fibril formation is very dependent on the incubation conditions, such as pH and stirring. Fibrils were observed at pH 2 with stirring, but not at pH 5.5 and 7.5. The conformation of the amyloid fibrils purified from patient organs were also measured, and the mass spectral characteristics of each of these and their proteolytic digests were also determined. Different experimental conditions are being tested for IgG light-chain fibril formation. The effects of the presence of glycosaminoglycans, e.g., heparin, heparin sulfate, during the fibril formation are also being explored, using highly sensitive and specific methods that have been developed by the Zaia group..

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 淀粉样原纤维由异常重折叠的蛋白质组成。 AFM 已证明其能够阐明体外原纤维形成过程。淀粉样蛋白如淀粉样蛋白和重组 IgG 轻链的原纤维形成已有报道。 Ionescu-Zanetti 等人提出的模型表明，淀粉样蛋白形成 ¿-片状结构，成为直径约为 2.4 nm 的单丝。两根或多根丝可以缠绕在一起直接形成较大尺寸的原纤维或原纤维。该 AFM 项目与我们的其他淀粉样蛋白质谱项目相关，重点研究从患者器官中纯化并重悬于溶液中的 IgG 轻链，以及直接取自人体器官的原纤维。 AL LC 原纤维悬浮在缓冲液中，并在不同时间取出等分试样，用于轻敲模式下的 AFM 分析。我们的初步数据表明，原纤维形成的速率很大程度上取决于孵育条件，例如 pH 值和搅拌。在搅拌下，在 pH 2 时观察到原纤维，但在 pH 5.5 和 7.5 时观察不到原纤维。还测量了从患者器官中纯化的淀粉样原纤维的构象，并测定了每种淀粉样原纤维及其蛋白水解消化物的质谱特征。正在测试 IgG 轻链原纤维形成的不同实验条件。糖胺聚糖（例如肝素、硫酸肝素）的存在对原纤维形成过程的影响也正在使用 Zaia 小组开发的高度灵敏和特定的方法进行探索。