AFM AND MS TO MONITOR FIBRIL FORMATION FROM AMYLOID IG LIGHT CHAINS AND GAGS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 淀粉样纤维是由异常折叠的蛋白质组成的。原子力显微镜已经证明了它能够解释体外纤维的形成过程。淀粉样蛋白如淀粉样蛋白和重组免疫球蛋白轻链的纤维形成已有报道。Ionescu-Zanetti等人提出的模型表明，淀粉样蛋白形成片状结构，形成直径约2.4 nm的单丝。两根或两根以上的细丝可以缠绕在一起，直接形成更大尺寸的原丝或原纤维。该AFM项目与我们的其他淀粉样质谱项目相关，重点是从患者器官提纯并在溶液中悬浮的免疫球蛋白轻链，以及直接从人体器官提取的纤维。在攻丝模式下，将Al LC纤维悬浮在缓冲液中，并在不同的时间取等分进行AFM分析。我们的初步数据表明，纤维形成的速度非常依赖于培养条件，如pH和搅拌。在pH=2时可观察到纤维，而在pH=5.5和7.5时未观察到纤维。还测量了从患者器官中提纯的淀粉样纤维的构象，并测定了每种淀粉样纤维及其蛋白水解物的质谱学特征。正在测试不同的实验条件以形成免疫球蛋白轻链纤维。使用Zaia小组开发的高度敏感和特殊的方法，也在探索糖胺多聚糖在纤维形成过程中的作用，例如肝素、肝素硫酸盐。

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..