AFM TO MONITOR FIBRIL FORMATION FROM AMYLOID IG LIGHT CHAINS

AFM 监测淀粉样蛋白 IG 轻链的纤维形成

• 批准号: 7723045
• 负责人: Vickery E Trinkaus-Randall
• 金额: $ 2.79万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723045
• 关键词: Acetates; Aliquot; Amyloid; Amyloid Fibrils; Amyloid Proteins; Buffers; Caliber; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Condition; Data; Filament; Funding; Glycosaminoglycans; Grant; Heparin; Human; Immunoglobulin G; In Vitro; Incubated; Inorganic Sulfates; Institution; Light; Mass Spectrum Analysis; Measures; Modeling; Molecular Conformation; Monitor; Organ; Patients; Process; Proteins; Rate; Recombinants; Reporting; Research; Research Personnel; Resources; Solutions; Source; Standards of Weights and Measures; Structure; Testing; Time; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; amyloid fibril formation; size; urinary

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. Then 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. Purified urinary IgG light chain protein (01-029) was incubated at 0.03 mM in the presence of buffer and 100 mM NaCl solution at 37 deg C with moderate stirring. Standard buffers included 20 mM HCl, 20 mM TRIS, and 50 mM acetate. Aliquots were 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, 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 will be tested for IgG light-chain fibril formation. The effects of glycosaminoglycan including heparin, heparin sulfate during the fibril formation are currently being explored.

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。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 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 已证明其能够阐明淀粉样蛋白（例如淀粉样蛋白）的原纤维形成过程。 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2.4 nm的单丝，然后两个或多个丝可以相互缠绕以直接形成更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，以及直接从人体器官中提取的纯化尿 IgG 轻链蛋白 (01-029)。 ）在缓冲液和 100 mM NaCl 溶液存在下在 37 ℃ 下以 0.03 mM 孵育，并适度搅拌标准缓冲液包括 20 mM HCl，在敲击模式下，在不同时间取出等份进行 AFM 分析，原纤维形成的速率很大程度上取决于孵育条件，例如在 pH 值、搅拌条件下观察到的原纤维。 pH 2 并搅拌，但不在 pH 5.5 和 7.5 下。还测量了从患者器官中纯化的淀粉样原纤维的构象，以及它们各自的质谱特征。还确定了它们的蛋白水解消化。将测试 IgG 轻链原纤维形成的不同实验条件。目前正在探索糖胺聚糖（包括肝素、硫酸肝素）在原纤维形成过程中的影响。