Division of Newborn Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Hum Mol Genet. 2021 Feb 25;29(24):3882-3891. doi: 10.1093/hmg/ddaa276.
Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.
横纹肌优先表达基因（SPEG）是肌球蛋白轻链激酶家族的成员，定位于骨骼肌肌原纤维周围三联体的水平。在人类中，SPEG 突变与中核肌病和心肌病有关。我们之前使用横纹肌特异性 Speg 敲除（KO）小鼠模型表明，SPEG 对三联体的维持和钙处理至关重要。在这里，我们进一步研究了 SPEG 的分子功能，并表征了 SPEG 缺乏对三联体和焦点粘连蛋白的影响。我们使用酵母双杂交测定法，鉴定出中间丝蛋白 desmin 与 SPEG 相互作用，并通过共免疫沉淀证实了这种相互作用。通过结构域映射测定法，我们定义了 SPEG 的 Ig 样和纤连蛋白 III 结构域与 desmin 的杆状结构域相互作用。在骨骼肌中，SPEG 耗竭导致体内 desmin 聚集体，并使 desmin 平衡从可溶性转移到不溶性部分。我们还使用 Western blot 和免疫荧光法分析了 Speg-KO 小鼠中三联体蛋白的表达和定位。RyR1 和 triadin 的量明显减少，而 DHPRα1、SERCA1 和 triadin 在 Speg-KO 肌纤维的离散区域异常积累。此外，Speg-KO 肌肉表现出肌动蛋白和整合素β1 的内化，它们都是焦点粘连复合物的关键组成部分。此外，整合素β1 在 Speg-KO 肌纤维的早期内体中异常积累。这些结果表明，SPEG 缺乏的骨骼肌表现出几种与 MTM1 缺乏相似的病理特征。共享细胞途径的缺陷可能是这两种疾病的结构和功能异常的基础。
