Zhao Junling, Kodippili Kasun, Yue Yongping, Hakim Chady H, Wasala Lakmini, Pan Xiufang, Zhang Keqing, Yang Nora N, Duan Dongsheng, Lai Yi
Department of Molecular Microbiology and Immunology, School of Medicine.
National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA.
Hum Mol Genet. 2016 Sep 1;25(17):3647-3653. doi: 10.1093/hmg/ddw210. Epub 2016 Jul 4.
Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. It has long been thought that membrane binding of dystrophin depends on its cysteine-rich (CR) domain. Here, we provide in vivo evidence suggesting that dystrophin contains three additional membrane-binding domains including spectrin-like repeats (R)1-3, R10-12 and C-terminus (CT). To systematically study dystrophin membrane binding, we split full-length dystrophin into ten fragments and examined subcellular localizations of each fragment by adeno-associated virus-mediated gene transfer. In skeletal muscle, R1-3, CR domain and CT were exclusively localized at the sarcolemma. R10-12 showed both cytosolic and sarcolemmal localization. Importantly, the CR-independent membrane binding was conserved in murine and canine muscles. A critical function of the CR-mediated membrane interaction is the assembly of the dystrophin-associated glycoprotein complex (DGC). While R1-3 and R10-12 did not restore the DGC, surprisingly, CT alone was sufficient to establish the DGC at the sarcolemma. Additional studies suggest that R1-3 and CT also bind to the sarcolemma in the heart, though relatively weak. Taken together, our study provides the first conclusive in vivo evidence that dystrophin contains multiple independent membrane-binding domains. These structurally and functionally distinctive membrane-binding domains provide a molecular framework for dystrophin to function as a shock absorber and signaling hub. Our results not only shed critical light on dystrophin biology and DMD pathogenesis, but also provide a foundation for rationally engineering minimized dystrophins for DMD gene therapy.
肌营养不良蛋白是一种大型的肌膜下蛋白。其缺失会导致杜兴氏肌营养不良症（DMD）。与肌膜结合对于肌营养不良蛋白保护肌肉免受收缩诱导损伤至关重要。长期以来，人们一直认为肌营养不良蛋白的膜结合依赖于其富含半胱氨酸的（CR）结构域。在此，我们提供体内证据表明肌营养不良蛋白包含另外三个膜结合结构域，包括血影蛋白样重复序列（R）1 - 3、R10 - 12和C末端（CT）。为了系统地研究肌营养不良蛋白的膜结合，我们将全长肌营养不良蛋白分成十个片段，并通过腺相关病毒介导的基因转移检查每个片段的亚细胞定位。在骨骼肌中，R1 - 3、CR结构域和CT仅定位于肌膜。R10 - 12显示出胞质和肌膜定位。重要的是，不依赖CR的膜结合在小鼠和犬类肌肉中是保守的。CR介导的膜相互作用的一个关键功能是肌营养不良蛋白相关糖蛋白复合物（DGC）的组装。虽然R1 - 3和R10 - 12不能恢复DGC，但令人惊讶的是，单独的CT就足以在肌膜上建立DGC。进一步的研究表明，R1 - 3和CT在心脏中也与肌膜结合，尽管相对较弱。综上所述，我们的研究提供了首个确凿的体内证据，表明肌营养不良蛋白包含多个独立的膜结合结构域。这些在结构和功能上独特的膜结合结构域为肌营养不良蛋白作为减震器和信号枢纽发挥作用提供了分子框架。我们的结果不仅为肌营养不良蛋白生物学和DMD发病机制提供了重要线索，也为合理设计用于DMD基因治疗的最小化肌营养不良蛋白奠定了基础。
