Probing the cellular Mechanisms of Transthyretin Cytotoxicity

描述（由申请人提供）：甲状腺素运载蛋白（TTR）淀粉样变性是一种致命的蛋白质二级结构疾病，其特征为来源于分泌型甲状腺素运载蛋白的聚集体（包括淀粉样纤维）的细胞外沉积。衰老是这些疾病的老年系统性（正常序列）和家族性（突变序列）形式的风险因素。此时，无可用的药物治疗。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是体内发病机制的关键。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）TTR在组织培养中的细胞毒性反映了人类病理学对靶组织的易感性和不同TTR变体的淀粉样蛋白原性。2)与年龄相关的蛋白质氧化增加与TTR淀粉样变性的发生之间存在关系。3)合适的TTR构象异构体和靶细胞之间的相互作用导致活性氧物质的产生，这反过来又触发导致细胞死亡的凋亡级联反应。4)存在介导细胞毒性反应的细胞受体。5)导致细胞死亡的一系列事件可以用结构上来源于植物多酚白藜芦醇和其他抗氧化剂的化合物来预防。这些假设将在我们的3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变体方面模拟人类病理学的组织培养系统。有了这些模型在手，我们将精确地确定负责TTR细胞毒性物种，并确定蛋白质氧化和细胞外基质对TTR诱导的细胞毒性的作用。在目标2中，我们将使用生物化学、免疫化学和蛋白质组学质谱来表征和鉴定参与细胞毒性反应的细胞受体。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，以发现可以在体内预防TTR淀粉样变性的物质。我们将确定活性小分子抑制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。 公共卫生相关性：甲状腺素运载蛋白淀粉样变性是一组与年龄相关的疾病，大多无法治疗，了解甚少，最终致命，影响美国数百万人和世界各地更多的人。我们将建立相关的组织培养模型，以确定甲状腺素运载蛋白损伤的细胞机制，并找到可用于治疗这些疾病的新化合物。这些发现将为治疗干预开辟新的途径，例如阻断甲状腺素运载蛋白毒性物质的形成，或抑制导致细胞死亡的细胞途径。

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. PUBLIC HEALTH RELEVANCE: The transthyretin amyloidoses are a set of age-related, mostly untreatable, poorly understood and ultimately fatal diseases affecting millions in the US and many more worldwide. We will create relevant tissue culture models to define the cellular mechanism of transthyretin damage and to find new compounds that can be used to treat these disorders. The findings will open new avenues for therapeutic intervention such as blockade of the formation of transthyretin toxic species, or inhibition of the cellular pathways that lead to cell death.