Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis
Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis
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DOI：
10.1016/j.jmb.2003.09.024
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发表时间：
2003-11-14
影响因子：
5.6
通讯作者：
Fontana, A
中科院分区：
文献类型：
--
作者：
de Laureto, PP;Taddei, N;Fontana, A
SH3结构域是由60-85个氨基酸残基组成的小蛋白质模块，存在于许多参与细胞内信号转导的蛋白质中。在酸性溶液中，牛磷脂酰肌醇3‘-激酶(P13-SH3)的p85α亚基的SH3结构域处于致密的变性状态，易于形成淀粉样纤维。已经发现，这种聚集过程基本上受溶液条件的调节。在这里，我们分析了P13-SH3的天然状态和酸变性状态的构象特征，分别用蛋白酶K和胃酶进行了有限的蛋白分解实验。此外，我们还分析了PI3-SH3在pH为1.2和2.0的聚集和淀粉样蛋白形成过程中不同阶段被胃酶水解的倾向，并将这些条件下的蛋白分解位点与天然和聚集的P13-SH3的构象特征进行了比较。结果表明，P13-SH3在低pH条件下形成的变性状态对蛋白质的降解具有较强的抵抗力，表明其处于部分折叠状态。连接天然蛋白质中的β链b和c的长环是这个结构中最容易发生蛋白质降解的区域。值得注意的是，在酸性溶液中最初由这种变性状态形成的P13-SH3聚集体显示出对长环蛋白降解的敏感性，这表明蛋白质在聚集的早期阶段变得更加展开。相比之下，在较长时间内形成的更明确的淀粉样蛋白纤维完全抵抗蛋白质降解。我们认为最初形成的蛋白质聚集体是相对动态的物种，能够很容易地重组它们的相互作用，从而形成非常有序的纤维结构。此外，早期聚集体中多肽链的无序和非天然特性可能在确定这些物种先前研究中所揭示的高细胞毒性方面具有重要意义。(C)2003爱思唯尔有限公司。保留所有权利。
The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidyl- inositol 3'-kinase (P13-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of P13-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated P13-SH3. The results demonstrate that the denatured state of P13-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of P13-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species. (C) 2003 Elsevier Ltd. All rights reserved.