Henderson Davin M, Lee Ann, Ervasti James M
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Proc Natl Acad Sci U S A. 2010 May 25;107(21):9632-7. doi: 10.1073/pnas.1001517107. Epub 2010 May 10.
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.
肌营养不良蛋白基因的突变通常通过蛋白质表达缺失导致杜氏肌营养不良症（DMD）。在一小部分患者中，错义突变可导致DMD、贝克肌营养不良症或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）中。为了验证ABD1错义突变通过损害肌动蛋白结合活性导致疾病的假说，我们将K18N、L54R、D165V、A168D、L172H和Y231N突变引入全长肌营养不良蛋白cDNA中，并对每种突变蛋白的生化特性进行了表征。K18N和L54R突变与人类最严重的疾病相关，且每种突变均导致肌动蛋白结合亲和力小幅但显著地下降了4倍，而其他四种突变蛋白的亲和力与野生型肌营养不良蛋白没有显著差异。更有趣的是，观察到野生型肌营养不良蛋白以单步、高度协同的方式展开。相比之下，所有六种突变蛋白都明显更容易发生热变性和聚集。我们的结果表明，ABD1中的错义突变可能都通过蛋白质不稳定性和聚集导致肌营养不良蛋白功能丧失，而不是通过配体结合功能丧失。然而，更严重的疾病进展可能是由于某些突变对蛋白质聚集和肌动蛋白结合活性受损的综合影响。
