Mechanisms of Aggregation in Light Chain Amyloidosis

描述（由申请方提供）：本提案的总体目标是鉴别由轻链淀粉样变性（AL）蛋白构成的错误折叠细胞毒性物质。AL是一种罕见的致命性错误折叠疾病，其特征是分泌免疫球蛋白轻链的单克隆浆细胞增殖，这些免疫球蛋白轻链错误折叠为重要器官中的淀粉样蛋白沉积物。目前的治疗减少了恶性单克隆浆细胞的数量。关于将可溶性蛋白质转化为不溶性AL原纤维的错误折叠过程以及在此过程中产生的毒性物质的性质知之甚少;识别毒性物质对于开发AL的靶向治疗至关重要。我们已经确定蛋白质AL-09形成改变的二聚体界面。单个体细胞突变负责二聚体界面的变化，导致热力学稳定性的丧失，并促进淀粉样蛋白相对于其种系（“野生型”）蛋白的形成。我们的初步数据显示，AL-09是我们实验室研究的最具淀粉样蛋白生成性的AL蛋白，其可溶性形式对心肌细胞具有高度毒性，并且其淀粉样纤维核心与改变的二聚体界面结构一致。此外，我们希望探索细胞毒性的替代机制，以解释在一些治疗后循环轻链显著减少但仍有疾病进展的患者中观察到的现象。基于这一信息，我们的中心假设是可溶性AL轻链种类具有高毒性。有不同的机制，产生铝有毒物质。AL轻链中的特异性体细胞突变导致蛋白质有利于一种或多种细胞毒性机制。在目标1中，我们将使用心肌细胞毒性试验和人小动脉扩张试验来检验蛋白质内化是细胞毒性所需的假设。对于目标2，我们将检验以下假设：某些AL原纤维的低热力学稳定性可能导致解聚，这可能在细胞毒性中发挥作用。此外，我们将通过检验以下假设来确定细胞毒性的替代机制：一些AL蛋白能够与免疫球蛋白分子的正常库中存在的非致病性轻链杂交。对于目标3，我们将检验以下假设：AL原纤维核心组成主要由每种AL蛋白占据的主要二聚体结构驱动，并将与蛋白质细胞毒性相关。我们的研究结果将产生集体影响，因为它们将通过识别常见的细胞毒性物质和AL原纤维核心区域，为AL的机制提供新的知识。这些知识对于开发合理的药物设计以最终消除这种疾病的破坏性后果至关重要。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to identify the misfolded cytotoxic species populated by light chain amyloidosis (AL) proteins. AL is a rare, fatal misfolding disease characterized by the proliferation of monoclonal plasma cells that secrete immunoglobulin light chains that misfold as amyloid deposits in vital organs. Current treatments reduce the population of the malignant monoclonal plasma cells. Little is known about the misfolding process converting soluble proteins into insoluble AL fibrils and the nature of the toxic species generated during this process; identifying the toxic species is crucial to developing targeted therapies for AL. We have determined that the protein AL-09 forms an altered dimer interface. A single somatic mutation is responsible for the change in the dimer interface, causing loss of thermodynamic stability, and promoting amyloid formation with respect to its germline ('wild type') protein. Our preliminary data show that AL-09 is the most amyloidogenic AL protein studied in our laboratory, it is highly toxic to cardiomyocytes in its soluble form, and its amyloid fibril core is consistent with the altered dimer interface structure. In addition, we want to explore alternative mechanisms of cytotoxicity that would explain the phenomenon observed with some patients who have achieved significant reduction of circulating light chain post-treatment but still have disease progression. Based on this information, our central hypothesis is that soluble AL light chain species are highly toxic. There are different mechanisms that generate AL toxic species. Specific somatic mutations in AL light chains cause the protein to favor one or more cytotoxic mechanisms. In aim 1, we will test the hypothesis that protein internalization is required for cytotoxicity using toxicity assays on cardiomyocytes and human arteriole dilation assays. For aim 2, we will test the hypothesis that low thermodynamic stability of some AL fibrils could cause disaggregation that may play a role in cytotoxicity. In addition, we will identify alternative mechanisms of cytotoxicity by testing the hypothesis that some AL proteins are able to cross seed with non-pathogenic light chains present in the normal repertoire of immunoglobulin molecules. For aim 3, we will test the hypothesis that AL fibril core composition is mostly driven by the predominant dimer structure populated by each AL protein and will correlate to protein cytotoxicity. Our results will have a collective impact because they will provide new knowledge into the mechanism of AL by identifying common cytotoxic species and AL fibril core regions. This knowledge will be essential for the development of rational drug design to ultimately eliminate the devastating consequences of this disease.