Probing the Biochemical Mechanism of Amyloid Diseases

探究淀粉样蛋白疾病的生化机制

• 批准号: 6771063
• 负责人: JEFFERY W KELLY
• 金额: $ 31.02万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6771063
• 关键词: X ray crystallography; amyloid proteins; amyloidosis; chimeric proteins; conformation; drug discovery /isolation; inhibitor /antagonist; intermolecular interaction; laboratory mouse; molecular assembly /self assembly; molecular pathology; pathologic process; protein biosynthesis; protein structure function; retinoid binding proteins; thyroid hormone binding protein; tissue /cell culture

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the biochemical mechanism of human amyloid disease, such that novel mechanism-based small molecule and macromolecular therapeutic strategies can be developed. It is now possible to prepare and isolate hybrid transthyretin (TTR) tetramers of defined subunit composition, reflecting those found in heterozygous familial amyloid polyneuropathy patients. In aim 1, the kinetics and thermodynamics of denaturation and amyloidogenicity of the hybrid tetramers will be evaluated as a function of added binding partners and a variety of physiologically relevant conditions to better understand the mechanistic features of amyloidogenesis. The feasibility of a trans-suppression approach for therapeutic intervention will be evaluated, and in related studies, the idea that only certain hybrid tetramers will be amenable to fibril formation at a given denaturation stress level, will be tested. A folded monomeric version of TTR enables the kinetics and thermodynamics of the amyloidogenic tertiary structural changes to be studied independent of the quaternary structural changes. Several hypotheses will be tested, including the idea that amyloid fibrils derived from different sequences have unique quaternary structures. A structure-based design approach for discovering small molecule inhibitors of TTR amyloid fibril formation will be expanded in aim 2 to develop bivalent inhibitors, in an effort to test the amyloid hypothesis in vitro, in cell lines and in a murine animal model in vivo. The kinetics of binding and dissociation of the best structurally diverse inhibitors will be evaluated to better understand their efficacy. In aim 3, the mechanistic connection between amyloidogenesis and the neuropathology characterizing these diseases will be sought. Efficient intralysosomal amyloid formation is observed in a macrophage cell line fed L55P TTR, but not WT TTR. The decreased viability of the cells converting soluble L55P into amyloid allows the use of expression profiling and protein analysis to understand how it is that amyloidosis reprograms the cell for demise. The idea that a rare cell secreting amyloid fibrils (or a lysed amyloid laden cell) could infect cells in its vicinity leading to the rapid onset of disease will be explored also.

描述（由申请人提供）：本研究的长期目标是 了解人类淀粉样蛋白疾病的生化机制， 基于机制的小分子和大分子治疗策略可以 开发现在可以制备和分离杂合甲状腺素运载蛋白（TTR） 确定的亚基组成的四聚体，反映了在 杂合子家族性淀粉样多发性神经病患者。在目标1中，动力学 以及杂合四聚体的变性和淀粉样变性的热力学 将作为添加的结合配偶体和各种 生理相关的条件，以更好地了解机制， 淀粉样变性的特征。反式抑制方法的可行性 在相关研究中， 只有某些杂合四聚体才能在 将测试给定的变性应力水平。折叠的单体版本 TTR使淀粉样蛋白生成的三级代谢的动力学和热力学 研究的结构变化独立于四级结构 变化几个假设将被测试，包括淀粉样蛋白， 源自不同序列的原纤维具有独特的四级结构。一 用于发现小分子抑制剂基于结构的设计方法 TTR淀粉样纤维的形成将在目标2中扩展，以形成二价 抑制剂，在努力测试淀粉样蛋白假说在体外，在细胞系 以及在鼠动物模型体内。结合和解离的动力学 最好的结构多样的抑制剂将进行评估，以更好地 了解其功效。在目标3中， 淀粉样蛋白生成和神经病理学表征这些疾病将是 寻找。在巨噬细胞中观察到有效的溶酶体内淀粉样蛋白形成 细胞系饲喂L55P TTR，但不饲喂WT TTR。细胞活力下降 将可溶性L55 P转化为淀粉样蛋白允许使用表达谱， 蛋白质分析来了解淀粉样变性是如何重新编程细胞的 死亡一种罕见的分泌淀粉样纤维的细胞（或溶解的淀粉样纤维） 淀粉样蛋白负载细胞）可以感染其附近的细胞，导致快速的 也将探讨疾病的发病情况。