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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 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的原纤维形成过程，例如淀粉样蛋白-¿ 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，两个或多个丝可以直接缠绕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，重点关注 IgG 轻链。从患者器官中纯化并重悬于溶液中，以及直接取自人体器官的原纤维悬浮于缓冲液中并在不同时间取出等分试样。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培养条件，例如在 pH 2 和搅拌下观察到原纤维的构象，但在 pH 5.5 和 7.5 时则观察不到原纤维的构象。还测量了从患者器官中纯化的淀粉样原纤维，并确定了每种淀粉样原纤维及其蛋白水解消化物的质谱特征，并正在测试 IgG 轻链的不同实验条件。纤维形成过程中糖胺聚糖（例如肝素、硫酸肝素）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。一份报告 AFM 结果的手稿。原纤维生长的电镜成像最近发表在《生物化学杂志》上。