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Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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-15) and peroxisome proliferator-activated receptor signalling (P < 9e-14).
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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-15) and peroxisome proliferator-activated receptor signalling (P < 9e-14).
CONCLUSIONS: 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.
摘要：
背景：SPEG（横纹肌优先表达蛋白激酶）中的常染色体隐性突变与伴有或不伴有扩张型心肌病（CNM5）的中央核肌病有关。SPEG的损失与有缺陷的三合会形成有关，异常激励-收缩耦合，钙处理不当和骨骼肌局灶性粘连复合物的破坏。为了阐明潜在的分子途径，我们利用多组学工具和分析来全面了解复杂的生物过程和分子功能。
方法：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（每个基因型n=4）以进行转录组学和质谱分析（WT为n=4；Speg-CKO小鼠为n=3）以进行蛋白质组学和磷酸蛋白质组学分析。此外,使用SPEG抗体对合并的肌肉裂解物(股四头肌，来自WT和Speg-CKO小鼠的腓肠肌和三头肌）。根据多组学结果，我们进行了实时定量PCR,免疫共沉淀和免疫印迹来验证结果。
结果：我们发现SPEG与Myospryn复合蛋白CMYA5，FSD2和RyR1相互作用，这对三联体的形成至关重要，SPEG缺乏会导致肌spryn复合物异常（CMYA5的蛋白质水平降低至22±3％[P<0.05]，FSD2的蛋白质水平降低至18±3％[P<0.01]）。此外，SPEG在S2902处磷酸化RyR1（在Speg-CKO小鼠中，在S2902处磷酸化水平降低至55±15％；P<0.05），并且其损失影响多个位点的JPH2磷酸化（T161的磷酸化增加[1.90±0.24倍]，S162[1.61±0.37倍]和S165[1.66±0.13倍]；S228和S231的磷酸化降低[39±6%]，S234[50±12%],S593[48±3％]和S613[66±10％]；S162的P<0.05，其他部位的P<0.01）。在分析转录组时，受SPEG缺乏影响的最多的通路包括细胞外基质-受体相互作用(P<1e-15)和过氧化物酶体增殖物激活受体信号传导(P<9e-14).
结论：我们已经阐明了SPEG在三合会中的关键作用，因为它与myospryn复合蛋白（CMYA5，FSD2和RyR1）密切合作，它调节RyR1中JPH2和S2902中各种残基的磷酸化水平，其缺乏与几种途径的失调有关。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各种遗传疾病中涉及的蛋白质分子功能的重要性。
方法：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（每个基因型n=4）以进行转录组学和质谱分析（WT为n=4；Speg-CKO小鼠为n=3）以进行蛋白质组学和磷酸蛋白质组学分析。此外,使用SPEG抗体对合并的肌肉裂解物(股四头肌，来自WT和Speg-CKO小鼠的腓肠肌和三头肌）。根据多组学结果，我们进行了实时定量PCR,免疫共沉淀和免疫印迹来验证结果。
结果：我们发现SPEG与Myospryn复合蛋白CMYA5，FSD2和RyR1相互作用，这对三联体的形成至关重要，SPEG缺乏会导致肌spryn复合物异常（CMYA5的蛋白质水平降低至22±3％[P<0.05]，FSD2的蛋白质水平降低至18±3％[P<0.01]）。此外，SPEG在S2902处磷酸化RyR1（在Speg-CKO小鼠中，在S2902处磷酸化水平降低至55±15％；P<0.05），并且其损失影响多个位点的JPH2磷酸化（T161的磷酸化增加[1.90±0.24倍]，S162[1.61±0.37倍]和S165[1.66±0.13倍]；S228和S231的磷酸化降低[39±6%]，S234[50±12%],S593[48±3％]和S613[66±10％]；S162的P<0.05，其他部位的P<0.01）。在分析转录组时，受SPEG缺乏影响的最多的通路包括细胞外基质-受体相互作用(P<1e-15)和过氧化物酶体增殖物激活受体信号传导(P<9e-14).
结论：我们已经阐明了SPEG在三合会中的关键作用，因为它与myospryn复合蛋白（CMYA5，FSD2和RyR1）密切合作，它调节RyR1中JPH2和S2902中各种残基的磷酸化水平，其缺乏与几种途径的失调有关。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各种遗传疾病中涉及的蛋白质分子功能的重要性。
