Mechanistic studies of transthyretin misfolding and amyloid formation through a c

描述（由申请人提供）：蛋白质错误折叠和淀粉样蛋白形成涉及许多疾病，如淀粉样蛋白病，朊病毒和阿尔茨海默病。朊病毒疾病的独特之处在于，天然折叠的朊病毒蛋白形成具有不同分子构象（朊病毒株）的聚集体，这是不同疾病表型的基础。1-3朊病毒株可能被编码在初级序列中，该蛋白的突变诱导不同的菌株，导致不同的疾病表型。最近的研究表明，菌株假说也适用于其他表现出不同疾病表型的淀粉样蛋白疾病。1,2,4,5非朊病毒淀粉样蛋白显示出广泛的构象多样性，这可能与表型变异有关。然而，人们对淀粉样蛋白不同的错误折叠途径和结构多样性的分子基础知之甚少。从天然状态到（部分）未展开的中间产物和最终产物淀粉样蛋白的初始转变的结构研究对于理解淀粉样蛋白多样性的分子机制至关重要。致病突变对错误折叠途径的影响也应加以研究。然而，由于与不同疾病表型相关的致病性突变数量有限，先前研究的淀粉样蛋白的综合生物物理研究一直具有挑战性。此外，研究最广泛的多肽，？-淀粉样蛋白和？-synuclein分别与阿尔茨海默病和帕金森病相关，是天然展开的，使得多肽不适合进行初始构象转变（错误折叠）的机制研究。本研究计划旨在利用溶液和固态核磁共振研究天然折叠蛋白转甲状腺素（TTR）的淀粉样蛋白形成机制。已知野生型和100多种TTR突变型的淀粉样蛋白形成可引起具有巨大表型多样性的各种淀粉样蛋白病本提案的主要假设是TTR的致病性突变形式可能具有不同的错误折叠途径，采用不同的淀粉样蛋白构象，具有不同的毒性活性，这可能导致不同的疾病表型和组织选择性沉积。该假设将通过研究天然折叠态到（部分）未折叠的淀粉样蛋白中间体的构象转变和淀粉样蛋白的结构表征来验证。特别是，具有创新标记方案的固态核磁共振将为淀粉样蛋白多样性提供有价值的见解。该提案的具体目的是探索：(1)天然TTR对淀粉样变性单体的错误折叠。(2)本地结构的变化？淀粉样蛋白形成过程中的结构。(3)突变对错误折叠途径和淀粉样蛋白结构的影响。了解错误折叠和淀粉样蛋白形成途径的机制对于制定有效的TTR淀粉样病治疗策略至关重要。

DESCRIPTION (provided by applicant): Protein misfolding and amyloid formation is implicated in numerous diseases such as amyloidoses, prion and Alzheimer's diseases. Prion disease is unique in that the natively folded prion protein forms aggregates with distinct molecular conformations (prion strains), which underlie different disease phenotypes.1-3 The prion strain may be encoded in the primary sequence and mutations of the protein induce different strains, causing distinct disease phenotypes. Recent studies have suggested the strain hypothesis is applicable to other amyloid diseases that also manifest diverse disease phenotypes.1,2,4,5 Nonprion amyloids were shown to exhibit a wide conformational diversity,6-9 which may be linked to the phenotype variations. However, little is known about molecular basis of the diverse misfolding pathways and structural diversity of amyloid. Structural studies of the initial transition from the native state to (partly) unfolded intermediate and the end product amyloid are essential to understanding molecular mechanism of amyloid diversity. Effect of the pathogenic mutations on misfolding pathway should also be examined. The comprehensive biophysical studies have, however, been challenging for previously investigated amyloidogenic proteins due to the limited number of pathogenic mutations associated with distinct disease phenotypes. In addition, the most extensively studied polypeptides, ?-amyloid and ?-synuclein associated with Alzheimer's and Parkinson's diseases respectively, are natively unfolded, rendering the polypeptides not amenable for mechanistic studies of the initial conformational transition (misfolding). This research program is aimed at investigating amyloid formation mechanisms of a natively folded protein, transthyretin (TTR), using both solution and solid-state NMR. Amyloid formation of wild type and more than 100 mutant forms of TTR are known to cause various amyloidoses with enormous phenotype diversity.10 The main hypothesis of this proposal is that pathogenic mutant forms of TTR may have distinct misfolding pathways, adopting diverse amyloid conformations with different toxic activities, which may result in diverse disease phenotypes and tissue-selective depositions. The hypothesis will be tested through the studies of conformational transition of the natively folded state to (partly) unfolded amyloidogenic intermediate and structural characterization of amyloid. In particular, solid-state NMR with innovative labeling schemes will provide valuable insights into amyloid diversity. Specific aims of the proposal are to explore: (1) Misfolding of the native TTR to amyloidogenic monomer. (2) Structural changes of the native ?-structure during amyloid formation. (3) Effect of the mutations on the misfolding pathway and amyloid structure. Mechanistic understanding of the misfolding and amyloid formation pathways would be critical to developing effective therapeutic strategies for TTR amyloidoses.