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其重要，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，并且在淀粉样蛋白组装的启动中起关键作用。我们研究了在 pH 7.4 下心脏淀粉样变性 AL 蛋白体外聚集的早期事件。在这项研究中，我们区分了一般聚集和淀粉样蛋白的形成。聚集是由单体的消失和通过透射电子显微镜 (TEM) 检测到我们称为非纤维状大分子 (NFM) 中间体的可沉淀中间体来定义的。淀粉样蛋白的形成是由单体的消失、硫黄素 T 荧光增强和 TEM 中存在的原纤维定义的。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。不同蛋白质之间的淀粉样蛋白形成和淀粉样蛋白产量是可变的。在静止阶段，所有蛋白质都表现出不同程度的解离。这些分离的物种可能在已经复杂的 AL 淀粉样变性病理生理学中发挥关键作用。解离程度与淀粉样蛋白的产量成反比。我们的结果强调了中间体/原纤维解离在 AL 淀粉样变性中的重要性和生理后果。