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与许多疾病如淀粉样变性、朊病毒和阿尔茨海默病有关。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的聚集体（朊病毒株），这是不同疾病表型的基础。1 -3朊病毒株可能在一级序列中编码，蛋白质的突变诱导不同的株，导致不同的疾病表型。最近的研究表明，应变假说适用于其他淀粉样疾病，也表现出不同的疾病表型。1，2，4，5非朊病毒淀粉样蛋白显示出广泛的构象多样性，6-9这可能与表型变异。然而，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）未折叠中间体和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。还应检查致病突变对错误折叠途径的影响。然而，由于与不同疾病表型相关的致病性突变数量有限，因此对先前研究的淀粉样蛋白的全面生物物理研究一直具有挑战性。此外，最广泛研究的多肽，？-淀粉样蛋白和？-分别与阿尔茨海默氏病和帕金森氏病相关的突触核蛋白是天然未折叠的，使得多肽不适合于初始构象转变（错误折叠）的机制研究。本研究计划的目的是调查淀粉样蛋白的天然折叠蛋白，甲状腺素运载蛋白（TTR）的形成机制，使用溶液和固态NMR。已知野生型和100多种TTR突变形式的淀粉样蛋白形成可引起各种淀粉样蛋白病，具有巨大的表型多样性。10该提案的主要假设是TTR的致病突变形式可能具有不同的错误折叠途径，采用具有不同毒性活性的不同淀粉样蛋白构象，这可能导致不同的疾病表型和组织选择性沉积。通过研究天然折叠状态到（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特征，将验证这一假设。特别是，固态NMR与创新的标记方案将提供有价值的见解淀粉样蛋白的多样性。该提案的具体目标是探索：（1）天然TTR错误折叠成淀粉样单体。(2)原住民的结构变化？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。理解错误折叠和淀粉样蛋白形成途径的机制对于开发TTR淀粉样变性的有效治疗策略至关重要。

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.