DYSF基因突变,编码蛋白质dyferlin,导致几种形式的肌营养不良。在健康的骨骼肌中,dysferlin集中在横小管中,并参与膜破裂后修复肌膜和稳定Ca2信号。DYSF基因编码7-8个C2结构域,几个Fer和Dysf域,和C端跨膜序列。因为它的编码序列太大,无法包装在腺相关病毒中,全长序列不适合当前的基因递送方法。因此,我们已经检查了更小版本的dysferlin,被称为“nanodysferlins,“旨在消除几个C2域,特别是C2域D,E,F;B,D,和E;和B,D,E,我们还通过替换Nanodysferlin缺失结构域D至F中C2G中的八个氨基酸产生了一个变体。我们用这些变体的金星融合构建体电穿孔了dysferlin-nullA/J小鼠肌纤维,或者作为未标记的纳米因子和GFP,标记转染的纤维我们发现,尽管这些nanodysferlins未能集中在横向小管中,其中三个支持激光损伤后的膜修复,而所有四个都结合了膜修复蛋白,TRIM72/MG53,类似于WTdysferlin。相比之下,在肌纤维受到轻度低渗性休克损伤后,它们未能抑制Ca2波。我们的结果表明,正常的t管定位和Ca2信号传导需要dysferlin的内部C2结构域,而膜修复不需要这些C2结构域。
Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed \"nanodysferlins,\" designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.