Mechanisms of Aggregation in Light Chain Amyloidosis

描述(由申请人提供)：我们提出的研究的总体目标是剖析导致轻链淀粉样变性(AL)沉积形成的分子机制。阿尔茨海默病是一种由血浆B细胞异常增殖引起的破坏性疾病。这些肿瘤细胞分泌单克隆性免疫球蛋白轻链，作为截断的轻链错误折叠成重要器官中的淀粉样沉积，导致组织损伤和器官衰竭。免疫球蛋白可变区中积累的体细胞突变使这些蛋白质的热力学稳定性低于它们的非淀粉样变性对应蛋白。免疫球蛋白轻链和重链都是在成熟的血浆B细胞中产生的，它们在那里结合形成抗体，并分泌到循环中。在大多数AL患者中，只有轻链被分泌到循环中，即使重链存在于浆细胞中，这表明无法与重链相关联。关于AL的许多问题仍然没有得到回答。目前尚不清楚是什么原因导致失去与重链的联系，导致蛋白质截断的细胞室或事件，以及截断是否是触发淀粉样蛋白形成的重要的不稳定事件。最后，鉴于AL具有丰富的突变多样性，还没有全面的研究来了解突变位置在AL的蛋白质折叠、稳定性和淀粉样变性中的作用。我们假设AL蛋白的不稳定是由于以下一个或多个事件引发的部分未折叠状态的采样：体细胞突变、从免疫球蛋白重链解离和/或蛋白水解性切割。这项工作的目标1将研究可变区、截短和全长AL蛋白的结构和稳定性。我们还将系统地将AL突变恢复到种系残基，以确定导致形成淀粉样蛋白倾向的突变。AIM 2将描述AL蛋白的淀粉样蛋白的形成。我们将研究硫酸钠稳定淀粉样蛋白生成中间体和促进淀粉样蛋白形成的作用。目的3研究AL的细胞生物学。我们将确定HC基因的完整性，AL蛋白与重链二聚的能力，并将研究细胞培养中轻链的内化，以确定是否发生了细胞内蛋白分解。这项工作将加深我们对AL发病机制的认识，有助于我们更好地描绘其治疗和治疗策略。

DESCRIPTION (provided by applicant): The overall goal of our proposed research is to dissect the molecular mechanisms that cause Light chain amyloidosis (AL) deposits to form. AL is a devastating disease caused by the abnormal proliferation of plasma B cells. These tumor cells secrete monoclonal immunoglobulin light chains that misfold as truncated light chains into amyloid deposits in vital organs, causing tissue damage and organ failure. Somatic mutations that accumulate in the immunoglobulin variable domain make these proteins less thermodynamially stable than their non-amyloidogenic counterparts. Both immunoglobulin light and heavy chains are produced in the mature plasma B cell where they associate to form antibodies that are secreted into circulation. In most AL patients, only light chain is secreted into circulation, even when the heavy chain is present in the plasma cell, suggesting a failure to associate with the heavy chain. Many questions remain unanswered in regards to AL. It is not clear what causes the loss of association with the heavy chain, the cellular compartment or event that leads to the truncation of the protein, and whether or not the truncation is an important destabilzing event that triggers amyloid formation. Finally, given the rich mutational diversity of AL, no comprehensive study has been done to understand the role of the location of mutations in protein folding, stability and amyloidogenicity in AL. We hypothesize that AL protein destabilization is due to sampling of partially unfolded states triggered by one or more of the following events: somatic mutations, dissociation from the immunoglobulin heavy chain, and/or proteolytic cleavage. Aim 1 of this work will study the structure and stability of variable domain, truncated and full length AL proteins. We will also perform a systematic restoration of AL mutations to germline residues to identify the mutations responsible for the propensity to form amyloid. Aim 2 will characterize amyloid formation by AL proteins. We will study the role of sodium sulfate stabilizing amyloidogenic intermediates and accelerating amyloid formation. Aim 3 will study the cell biology of AL. We will determine the integrity of the HC gene, the ability of AL proteins to dimerize with heavy chains and will study internalization of light chains in cell culture to determine if intracellular proteolysis is occurring. This work will increase our understanding of the mechanism of AL pathogenesis, helping us delineate better strategies for its management and cure.