Laboratory of Bio-otology, Department of Otolaryngology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Int J Mol Sci. 2023 Feb 28;24(5):4707. doi: 10.3390/ijms24054707.
Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair.
定量表面等离子体共振（SPR）用于确定与肢带型肌营养不良 2B/R2 中断的可能介导骨骼肌修复的蛋白之间的 dysferlin 直接相互作用的结合强度和钙依赖性。dysferlin 经典 C2A（cC2A）和 C2F/G 结构域直接与膜联蛋白 A1、钙蛋白酶-3、 caveolin-3、affixin、AHNAK1、突触素-4 和 mitsugumin-53 相互作用，cC2A 是主要靶点，C2F 参与较少，总体表现出正钙依赖性。在几乎所有情况下，dysferlin C2 配对仅表现出负钙依赖性。与 otoferlin 一样，dysferlin 通过其羧基末端与 FKBP8（一种抗凋亡的外线粒体膜蛋白）直接相互作用，并通过其 C2DE 结构域与凋亡相关基因（ALG-2/PDCD6）相互作用，将抗凋亡与凋亡联系起来。共聚焦 Z 堆叠免疫荧光证实了 PDCD6 和 FKBP8 在肌膜上的共区室化。我们的证据支持这样的假设，即在损伤之前，dysferlin C2 结构域自我相互作用，并产生一个折叠的、紧凑的结构，如 otoferlin 所示。随着损伤时细胞内 Ca 的升高，dysferlin 将展开并暴露 cC2A 结构域以与膜联蛋白 A1、钙蛋白酶-3、mitsugumin 53、affixin 和 caveolin-3 相互作用，并且 dysferlin 将从其与 PDCD6 的相互作用重新排列到基础钙水平以与 FKBP8 强烈相互作用，这是一种促进膜修复的分子内重排。
