Division of Neonatology, Department of Pediatrics, University of Miami Miller School of Medicine and Holtz Children's Hospital, Jackson Health System, Miami, FL, USA.
Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
J Cachexia Sarcopenia Muscle. 2024 Jun;15(3):1003-1015. doi: 10.1002/jcsm.13470. Epub 2024 May 9.
Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions.
Skeletal muscles from 2-month-old SPEG-deficient (Speg-CKO) and wild-type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg-CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg-CKO mice. Based on the multi-omics results, we performed quantitative real-time PCR, co-immunoprecipitation and immunoblot to verify the findings.
We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg-CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24-fold], S162 [1.61 ± 0.37-fold] and S165 [1.66 ± 0.13-fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P < 1e) and peroxisome proliferator-activated receptor signalling (P < 9e).
We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG-interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi-omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.
常染色体隐性突变 SPEG（横纹肌优先表达蛋白激酶）与伴或不伴扩张型心肌病的核性肌病 5 型（CNM5）有关。SPEG 的缺失与三联体形成缺陷、兴奋-收缩偶联异常、钙处理异常和骨骼肌粘着斑复合物破坏有关。为了阐明潜在的分子途径，我们利用多组学工具和分析方法，全面了解复杂的生物学过程和分子功能。
使用 2 个月大的 SPEG 缺陷（Speg-CKO）和野生型（WT）小鼠的骨骼肌进行 RNA 测序（每组 4 只）以分析转录组学，以及进行质谱分析（WT 组 4 只，Speg-CKO 组 3 只）以分析蛋白质组学和磷酸化蛋白质组学。此外，还使用 SPEG 抗体在 WT 和 Speg-CKO 小鼠的混合肌肉裂解物（股四头肌、比目鱼肌和三头肌）上进行相互作用组学分析。基于多组学结果，我们进行了定量实时 PCR、共免疫沉淀和免疫印迹实验以验证发现。
我们发现 SPEG 与三联体形成关键的肌联蛋白复合物蛋白 CMYA5、FSD2 和 RyR1 相互作用，并且 SPEG 缺乏会导致肌联蛋白复合物异常（CMYA5 的蛋白水平降低至 22 ± 3% [P < 0.05]，FSD2 降低至 18 ± 3% [P < 0.01]）。此外，SPEG 在 S2902 处磷酸化 RyR1（Speg-CKO 小鼠中的 S2902 磷酸化水平降低至 55 ± 15%；P < 0.05），其缺失会影响 JPH2 在多个位点的磷酸化（T161 处的磷酸化增加[1.90 ± 0.24 倍]，S162 处增加[1.61 ± 0.37 倍]，S165 处增加[1.66 ± 0.13 倍]；S228 和 S231 处的磷酸化减少[39 ± 6%]，S234 处减少[50 ± 12%]，S593 处减少[48 ± 3%]，S613 处减少[66 ± 10%]；S162 处 P < 0.05，其他位点 P < 0.01）。在分析转录组时，受 SPEG 缺乏影响最严重的失调途径包括细胞外基质-受体相互作用（P < 1e）和过氧化物酶体增殖物激活受体信号通路（P < 9e）。
我们已经阐明了 SPEG 在三联体中的关键作用，因为它与肌联蛋白复合物蛋白（CMYA5、FSD2 和 RyR1）密切相关，它调节 JPH2 和 RyR1 中各种残基的 S2902 磷酸化水平，并且其缺乏与几个途径的失调有关。该研究确定了独特的 SPEG 相互作用蛋白及其磷酸化功能，并强调了使用多组学方法全面评估涉及各种遗传疾病的蛋白质的分子功能的重要性。
