Mechanisms of Aggregation in Light Chain Amyloidosis

描述（由申请人提供）：本提案的总体目标是鉴定由轻链淀粉样变性（AL）蛋白填充的错误折叠的细胞毒性物种。AL是一种罕见的、致命的错误折叠疾病，其特征是单克隆浆细胞增殖，分泌免疫球蛋白轻链，在重要器官中错误折叠为淀粉样蛋白沉积物。目前的治疗方法减少了恶性单克隆浆细胞的数量。对于可溶性蛋白转化为不溶性AL原纤维的错误折叠过程以及在此过程中产生的有毒物质的性质知之甚少；确定有毒物质对于开发针对AL的靶向治疗至关重要。我们已经确定AL-09蛋白形成了一个改变的二聚体界面。单个体细胞突变可导致二聚体界面的变化，导致热力学稳定性的丧失，并促进其种系（“野生型”）蛋白的淀粉样蛋白形成。我们的初步数据表明，AL-09是我们实验室研究的最具淀粉样蛋白，其可溶性形式对心肌细胞具有高毒性，其淀粉样纤维核心与改变后的二聚体界面结构一致。此外，我们希望探索细胞毒性的其他机制，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。基于这些信息，我们的中心假设是可溶性AL轻链物种是剧毒的。产生AL毒性物质的机制不同。AL轻链的特异性体细胞突变导致蛋白质倾向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验证蛋白质内化是细胞毒性所必需的假设。对于目的2，我们将检验一些AL原纤维的低热力学稳定性可能导致分解的假设，这可能在细胞毒性中起作用。此外，我们将通过验证一些AL蛋白能够与存在于正常免疫球蛋白分子库中的非致病性轻链杂交的假设来确定细胞毒性的替代机制。对于目标3，我们将验证AL原纤维核心组成主要由每个AL蛋白填充的主要二聚体结构驱动的假设，并将与蛋白质的细胞毒性相关。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种和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.