PARTIAL DENATURATION OF TRANSTHYRETIN IS SUFFICIENT FOR AMYLOID FIBRIL FORMATION INVITRO
PARTIAL DENATURATION OF TRANSTHYRETIN IS SUFFICIENT FOR AMYLOID FIBRIL FORMATION INVITRO
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DOI：
10.1021/bi00151a036
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发表时间：
1992-09-15
期刊：
影响因子：
2.9
通讯作者：
KELLY, JW
中科院分区：
文献类型：
--
作者：
COLON, W;KELLY, JW
淀粉样蛋白疾病是由给定的蛋白质自组装成不溶的交叉β-折叠的四元结构形式引起的，这是致病的。了解淀粉样蛋白原纤维形成的生化机制将有助于了解淀粉样蛋白疾病。为此，已经开发了一种在模拟溶酶体的酸性环境的条件下将淀粉样蛋白转甲状腺蛋白转化为淀粉样纤维的方法。在体外，结构性的跨甲状腺维甲素变性中间体的结合足以形成淀粉样原纤维。纤维形成的速度依赖于pH，在溶酶体内可达的PHS处观察到显著的速度(3.6-4.8)。远紫外圆二色谱研究表明，在形成纤维的PHS处，转甲腺激素保留了其二级结构特征。近紫外圆二色谱研究表明，在纤维形成过程中，转甲状腺维A酸也保留了大部分的三级结构。近紫外CD分析结合戊二醛交联研究表明，在发生纤维形成的pH范围内，pH介导的四聚体到单体的转变是有效的。当pH值低于3.6时，纤维形成速率显著降低，这与变性为单体TTR中间体一致，该中间体失去了天然的三级结构和形成纤维的能力。目前还很难确定哪种四级结构形式的转甲状腺素是淀粉样变性中间体。这些困难的出现是因为在pH 3.6时纤维形成的速率最大，此时观察到四聚体、微量的二聚体和大量的单体。芳香族紫外光分析表明，存在一个以pH 5.3为中心的微妙的四聚体重排，这表明与淀粉样纤维相关的绝对不是天然的四聚体。光谱研究和交联实验表明，淀粉样蛋白前体为后向四聚体或单体中间体。这些研究表明，TTR可以在与溶酶体相似的酸性条件下自组装成跨甲状腺维甲素淀粉样纤维，支持溶酶体和/或内体参与淀粉样病的可行性。
Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed. Aromatic UV analysis demonstrates the presence of a subtle tetramer rearrangement which is centered around pH 5.3, indicating that it is definitely not the native tetramer that associates into amyloid fibrils. Spectroscopic studies and cross-linking experiments are consistent with either a rear ranged tetramer or a monomeric intermediate as the amyloid precursor. These studies demonstrate that TTR can self-assemble into transthyretin amyloid fibrils under acidic conditions similar to those found in a lysosome, supporting the feasibility of lysosomal and/or endosomal involvement in amyloid disease.