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: 8365539
• 负责人: Vickery E Trinkaus-Randall
• 金额: $ 1.54万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365539
• 关键词: Aliquot; Amyloid; Amyloid Fibrils; Amyloid Proteins; Biochemistry; Biology; Buffers; Caliber; Characteristics; Data; Filament; Funding; Gagging; Glycosaminoglycans; Grant; Growth; Heparin; Human; Image; Immunoglobulin G; In Vitro; Inorganic Sulfates; Journals; Light; Manuscripts; Mass Spectrum Analysis; Measures; Medicine; Methods; Modeling; Molecular Conformation; Monitor; National Center for Research Resources; Organ; Patients; Principal Investigator; Process; Proteins; Publishing; Recombinants; Reporting; Research; Research Infrastructure; Resources; Solutions; Source; Structure; Testing; Time; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; amyloid fibril formation; cost

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. A manuscript that reports the results from AFM and EM imaging of fibril growth was recently published in the Journal of Biological Chemistry.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 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。两个或多个细丝可以交织以直接形成较大尺寸的协议或原纤维。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。 Al LC原纤维被悬浮在缓冲液中，并在敲击模式下在不同时间进行AFM分析。我们的初步数据表明，原纤维形成的速率非常取决于孵育条件，例如pH和搅拌。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。正在测试不同的实验条件的IgG轻链纤维形成。还探索了ZAIA组开发的高度敏感和特定的方法，在原纤维形成期间也探索了乙酰氨基聚糖，例如肝素，硫酸肝素硫酸盐的影响。一份报告了AFM的结果和原纤维生长成像的手稿，最近发表在《生物化学杂志》上。