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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 淀粉样蛋白原纤维由绝对重折叠的蛋白质组成。 AFM已经证明了其阐明体外原纤维形成过程的能力。已经报道了淀粉样蛋白的原纤维形成，例如淀粉样蛋白和重组IgG轻链。 Ionescu -Zanetti等人提出的模型表明，淀粉样蛋白形成 - 表结构，成为直径约2.4 nm的单细丝。然后，两个或多个丝可以交织在一起以直接形成较大的尺寸协议或原纤维。 该AFM项目与我们的其他淀粉样蛋白质谱项目有关，并着重于从患者器官中纯化并恢复溶液中的IgG轻链，以及直接从人体器官中获得的原纤维。纯化的尿液IgG轻链蛋白（01-029）在缓冲液和100 mM NaCl溶液的存在下以0.03 mm的孵育在37 g c下进行中等搅拌。标准缓冲液包括20毫米HCl，20毫米Tris和50毫米乙酸盐。在敲击模式下，在不同时间进行AFM分析的等分试样。我们的初步数据表明，原纤维形成的速率非常取决于孵化条件，例如pH，搅拌。搅拌时在pH 2处观察到原纤维，但在pH 5.5和7.5时观察到了原纤维。还测量了从患者器官纯化的淀粉样蛋白原纤维的组成，并且还确定了每个淀粉样蛋白的质谱特征及其蛋白水解消化。不同的实验条件将测试IgG轻链纤维形成。目前正在探索包括肝素，硫酸肝素硫酸盐在原纤维形成过程中的糖胺聚糖的作用。