Abstract

Early events in immunoglobulin light chain (AL) amyloid formation are especially important as some early intermediates formed during the aggregation reaction are cytotoxic and play a critical role in the initiation of amyloid assembly. We investigated the early events in in vitro aggregation of cardiac amyloidosis AL proteins at pH 7.4. In this study we make distinctions between general aggregation and amyloid formation. Aggregation is defined by the disappearance of monomers and the detection of sedimentable intermediates we call non-fibrillar macromolecular (NFM) intermediates by transmission electron microscopy (TEM). Amyloid formation is defined by the disappearance of monomers, Thioflavin T fluorescence enhancement, and the presence of fibrils by TEM. All proteins aggregated at very similar rates via the formation of NFM intermediates. The condensed NFM intermediates were composed of non-native monomers. Amyloid formation and amyloid yield was variable among the different proteins. During the stationary phase, all proteins demonstrated different degrees of dissociation. These dissociated species could play a key role in the already complex pathophysiology of AL amyloidosis. The degree of dissociation is inversely proportional to the amyloid yield. Our results highlight the importance and physiological consequences of intermediates/fibril dissociation in AL amyloidosis.

摘要

免疫球蛋白轻链（AL）淀粉样蛋白形成的早期事件尤为重要，因为在聚集反应过程中形成的一些早期中间体具有细胞毒性，并在淀粉样蛋白组装的启动中起关键作用。我们调查了心脏淀粉样变性AL蛋白在pH 7.4体外聚集的早期事件。在这项研究中，我们区分一般聚集和淀粉样蛋白形成。聚集是由单体的消失和可沉积的中间体的检测定义的，我们通过透射电子显微镜（TEM）将其称为非原纤维大分子（NFM）中间体。淀粉样蛋白的形成由单体的消失，硫黄素T荧光增强和TEM的原纤维存在来定义。所有蛋白质以非常相似的速率聚集通过形成NFM中间体。缩合的NFM中间体由非天然单体组成。淀粉样蛋白的形成和淀粉样蛋白的产量在不同蛋白质之间是可变的。在固定阶段，所有蛋白质均表现出不同程度的解离。这些解离的物种可能在AL淀粉样变性病已经很复杂的病理生理学中起关键作用。解离度与淀粉样蛋白产量成反比。我们的研究结果突出了AL淀粉样变性中中间体/原纤维解离的重要性和生理后果。