Probing the Biochemical Mechanism of Amyloid Diseases

描述(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，如此新颖 基于机理的小分子和大分子治疗策略可以 发展起来的。现在有可能制备和分离杂交反式甲状腺激素(TTR)。 定义的亚基组成的四聚体，反映在 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 杂化四聚体变性和淀粉样变性的热力学 将根据添加的绑定伙伴和各种 生理相关条件，以更好地理解 淀粉样变的特点。反式抑制方法的可行性 将对治疗干预进行评估，在相关研究中， 认为只有某些杂化四聚体才能在 一个给定的变性压力水平，将被测试。折叠的单体版本 使淀粉样变性三级结构的动力学和热力学成为可能 独立于第四纪构造研究的构造变化 改变。将测试几种假说，包括淀粉样蛋白 来自不同序列的纤维具有独特的四元结构。一个 基于结构的设计方法发现小分子抑制剂 目标2中将扩大Ttr淀粉样原纤维的形成以形成二价体 在体外，在细胞系中测试淀粉样蛋白假说的抑制剂 在活体的小鼠动物模型中。结合和解离的动力学 最好的结构多样化的抑制剂将被评估为更好 了解它们的功效。在目标3中，机构之间的联系 淀粉样变和以这些疾病为特征的神经病理学将是 被追寻。巨噬细胞内有效的溶酶体内淀粉样蛋白形成。 细胞系给予L55P TTR，但不给予WT TTR。细胞活力的下降 将可溶性L55P转化为淀粉样蛋白允许使用表达谱和 蛋白质分析以了解淀粉样变性是如何对细胞重新编程的 为死亡而死。一种罕见的分泌淀粉样纤维(或裂解的淀粉样纤维)的细胞 可感染其附近的细胞，导致快速的 还将探讨疾病的发病情况。

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.