Probing the cellular Mechanisms of Transthyretin Cytotoxicity

描述(申请人提供)：转甲状腺素(TTR)淀粉样变性是一种致命性的次级蛋白质结构障碍，其特征是由分泌的转甲状腺激素蛋白产生的聚集体，包括淀粉样纤维在细胞外沉积。衰老是这些疾病的老年性全身性(正常序列)和家族性(突变序列)形式的危险因素。目前，还没有可用的药物治疗。最近的数据表明，在TTR错误折叠过程中早期产生的寡聚体可能在体内的发病机制中起关键作用。我们已经开发了细胞和分子生物学工具来精确分析细胞损伤的分子机制。这些知识对于寻找治疗这些疾病的新治疗策略的长期目标至关重要。我们的工作假设是：1)组织培养中的TTR细胞毒性反映了关于靶组织的易感性和不同TTR变异体的淀粉样变性的人类病理。2)与年龄相关的蛋白质氧化增加与TTR淀粉样变性的发病有关。3)合适的TTR构象与靶细胞之间的相互作用导致活性氧物种的产生，进而触发导致细胞死亡的凋亡级联反应。4)存在细胞受体，介导细胞毒性反应。5)从植物多酚白藜芦醇和其他抗氧化剂中结构衍生的化合物可以防止导致细胞死亡的一系列事件。这些假设将在我们的三个具体目标中得到检验。在目标1中，我们将建立在组织特异性和TTR变异体方面模拟人类病理的组织培养系统。有了这些模型，我们将准确地确定负责的TTR细胞毒性物种，并确定蛋白质氧化和细胞外基质在TTR诱导的细胞毒性中的作用。在目标2中，我们将使用生化、免疫化学和蛋白质组质谱来表征和鉴定参与细胞毒反应的细胞受体。在目标3中，我们将使用细胞系统来筛选抑制TTR诱导的细胞毒性的化合物，以寻找可能在体内预防TTR淀粉样变性的物质。我们将明确活性小分子抑制TTR诱导的细胞毒性的机制，这反过来可能适用于其他淀粉样变性。从这些研究中获得的见解将为治疗干预提供先导化合物和潜在的新途径。

DESCRIPTION (provided by applicant): The transthyretin (TTR) amyloidoses are fatal disorders of secondary protein structure characterized by extracellular deposition of aggregates, including amyloid fibrils, derived from the secreted transthyretin protein. Aging is a risk factor for both the senile systemic (normal sequence) and familial (mutated sequence) forms of these disorders. At this time, no pharmacologic treatment is available. Recent data suggest that oligomers generated early in the course of TTR misfolding may be critical in the in vivo pathogenesis. We have developed the cell and molecular biologic tools to precisely analyze the molecular mechanisms underlying cell damage. Such knowledge is critical to the long-term goal of finding new therapeutic strategies to treat these diseases. Our working hypotheses are 1) TTR cytotoxicity in tissue culture reflects the human pathology regarding the susceptibility of target tissues and the amyloidogenicity of different TTR variants. 2) There is a relationship between the age-related increase in protein oxidation and the onset of TTR amyloidoses. 3) The interaction between the appropiate TTR conformer and target cells results in the generation of reactive oxygen species which, in turn, trigger an apoptotic cascade leading to cell death. 4) There is a cell receptor mediating the cytotoxic response. 5) The sequence of events leading to cell death can be prevented with compounds structurally derived from the plant polyphenol resveratrol and other antioxidants. These hypotheses will be tested in our 3 specific aims. In aim 1 we will establish tissue culture systems that mimic the human pathology in terms of tissue specificity and TTR variant. With these models in hand we will precisely identify the responsible TTR cytotoxic species, and define the role of protein oxidation and of the extracellular matrix on TTR-induced cytotoxicity. In aim 2 we will characterize and identify the cell receptor involved in the cytotoxic response using biochemical, immunochemical and proteomic mass spectrometry. In aim 3 we will use the cell system to screen compounds that inhibit TTR-induced cytotoxicity to find substances that may prevent TTR amyloidosis in vivo. We will define the mechanism of inhibition of TTR- induced cytotoxicity by the active small molecules which might in turn, be applicable to other amyloidoses. The insights gained from these studies will provide lead compounds and potential new pathways for therapeutic intervention.