Disease-causing missense mutations in actin binding domain 1 of dystrophin induce thermodynamic instability and protein aggregation
Disease-causing missense mutations in actin binding domain 1 of dystrophin induce thermodynamic instability and protein aggregation
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DOI：
10.1073/pnas.1001517107
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发表时间：
2010-05-25
影响因子：
11.1
通讯作者：
Ervasti, James M.
中科院分区：
文献类型：
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作者：
Henderson, Davin M.;Lee, Ann;Ervasti, James M.
肌营养不良蛋白基因的突变导致杜氏肌营养不良症（DMD）最常见的是通过蛋白质表达的丧失。在一小群患者中，错义突变可导致DMD、贝克尔肌营养不良症或X连锁心肌病。近一半的致病错义突变位于肌营养不良蛋白的肌动蛋白结合结构域1（ABD1）。为了检验ABD1错义突变通过损害肌动蛋白结合活性而引起疾病的假设，我们将K18 N、L54 R、D165 V、A168 D、L172 H和Y231 N突变工程化到全长肌营养不良蛋白cDNA中，并表征每种突变蛋白的生化特性。K18 N和L54 R突变与人类中最严重的疾病相关，并且各自引起肌动蛋白结合亲和力的小但显著的4倍降低，而其他四种突变蛋白的亲和力与WT肌营养不良蛋白没有显著差异。更有趣的是，观察到WT肌养蛋白以单步、高度合作的方式展开。相比之下，所有六种突变体蛋白质明显更易于热变性和聚集。我们的研究结果表明，ABD1的错义突变可能都通过蛋白质的不稳定性和聚集而不是通过配体结合功能的丧失导致肌营养不良蛋白功能的丧失。然而，更严重的疾病进展可能是由于一些突变对蛋白质聚集和受损的肌动蛋白结合活性的组合效应。
Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) most commonly through loss of protein expression. In a small subpopulation of patients, missense mutations can cause DMD, Becker muscular dystrophy, or X-linked cardiomyopathy. Nearly one-half of disease-causing missense mutations are located in actin-binding domain 1 (ABD1) of dystrophin. To test the hypothesis that ABD1 missense mutations cause disease by impairing actin-binding activity, we engineered the K18N, L54R, D165V, A168D, L172H, and Y231N mutations into the full-length dystrophin cDNA and characterized the biochemical properties of each mutant protein. The K18N and L54R mutations are associated with the most severe diseases in humans and each caused a small but significant 4-fold decrease in actin-binding affinity, while the affinities of the other four mutant proteins were not significantly different from WT dystrophin. More interestingly, WT dystrophin was observed to unfold in a single-step, highly cooperative manner. In contrast, all six mutant proteins were significantly more prone to thermal denaturation and aggregation. Our results suggest that missense mutations in ABD1 may all cause loss of dystrophin function via protein instability and aggregation rather than through loss of ligand binding function. However, more severe disease progressions may be due to the combinatorial effects of some mutations on both protein aggregation and impaired actin-binding activity.