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。AL是一种由血浆B细胞异常增殖引起的毁灭性疾病。这些肿瘤细胞分泌单克隆免疫球蛋白轻链，这些单克隆免疫球蛋白轻链作为截断的轻链错误折叠成淀粉样蛋白沉积在重要器官中，导致组织损伤和器官衰竭。体细胞突变在免疫球蛋白可变结构域的积累使得这些蛋白的热力学稳定性低于它们的非淀粉样蛋白对应体。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，在那里它们结合形成抗体，分泌到循环中。在大多数AL患者中，即使浆细胞中存在重链，也只有轻链分泌到循环中，这表明与重链的关联失败。关于AL的许多问题仍未得到解答。目前尚不清楚是什么原因导致与重链、细胞室或导致蛋白质截短的事件的关联丧失，以及截短是否是触发淀粉样蛋白形成的重要不稳定事件。最后，由于AL具有丰富的突变多样性，目前还没有全面的研究来了解突变位置在AL蛋白折叠、稳定性和淀粉样变性中的作用。我们假设AL蛋白不稳定是由于以下一种或多种事件触发的部分未折叠状态的采样：体细胞突变、免疫球蛋白重链解离和/或蛋白水解裂解。本工作的目的1将研究可变结构域、截断和全长AL蛋白的结构和稳定性。我们还将进行AL突变到种系残基的系统恢复，以确定导致淀粉样蛋白形成倾向的突变。目的2将描述AL蛋白淀粉样蛋白的形成。我们将研究硫酸钠稳定淀粉样蛋白中间体和加速淀粉样蛋白形成的作用。目的3将研究AL的细胞生物学。我们将确定HC基因的完整性，AL蛋白与重链二聚体的能力，并将研究细胞培养中轻链的内化，以确定细胞内蛋白水解是否发生。这项工作将增加我们对AL发病机制的理解，帮助我们描绘出更好的管理和治疗策略。

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.