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: 8170907
• 负责人: Vickery E Trinkaus-Randall
• 金额: $ 1.11万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170907
• 关键词: Aliquot; Amyloid; Amyloid Fibrils; Amyloid Proteins; Biochemistry; Buffers; Caliber; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Data; Filament; Funding; Gagging; Glycosaminoglycans; Grant; Growth; Heparin; Human; Image; Immunoglobulin G; In Vitro; Inorganic Sulfates; Institution; Journals; Light; Manuscripts; Mass Spectrum Analysis; Measures; 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. A manuscript that reports the results from AFM and EM imaging of fibril growth is under review by the Journal of Biological Chemistry.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 淀粉样原纤维由异常重折叠的蛋白质组成。原子力显微镜已经证明了它的能力，阐明在体外原纤维的形成过程。淀粉样蛋白如淀粉样蛋白和重组IgG轻纤维的形成链被举报。 由Ionescu-Zanetti等人提出的模型表明，淀粉样蛋白形成<$-sheet结构，成为直径约2.4 nm的单丝。两条或两条以上的丝状体可直接交织形成较大的原丝或原纤维。这个AFM项目与我们的其他淀粉样蛋白质谱项目有关，重点是从患者器官中纯化并重新悬浮在溶液中的IgG轻链，以及直接从人体器官中提取的原纤维。将AL LC原纤维悬浮在缓冲液中，并在不同时间取等分试样用于轻敲模式下的AFM分析。我们的初步数据表明，原纤维形成的速率非常依赖于孵育条件，如pH和搅拌。在pH 2下搅拌观察到原纤维，但在pH 5.5和7.5下未观察到原纤维。还测量了从患者器官纯化的淀粉样蛋白原纤维的构象，并且还确定了这些中的每一个及其蛋白水解酶的质谱特征。正在测试IgG轻链原纤维形成的不同实验条件。糖胺聚糖的存在的影响，例如， 肝素，硫酸肝素，在原纤维形成过程中的作用也在探索中，使用Zaia小组开发的高度敏感和特异的方法。一份报告原纤维生长的AFM和EM成像结果的手稿正在接受《生物化学杂志》的审查。