MSTN-/-牛肌肉泛素化蛋白组学分析及泛素化途径对牛成肌分化的调控作用机制研究

Ubiquitination is a key post-translational modification and plays an important role in many biological processes. In our previous study, muscle proteomic analysis of MSTN-/- cattle and wild-type cattle was conducted, and two important ubiquitinating regulatory enzymes PSMB5 (Proteasome subunit beta type-5) and UCH-L3 (Ubiquitin carboxyl-terminal hydrolase isozyme L3) were found significantly differentially expressed between MSTN-/- cattle and wild-type cattle. Our preliminary study reviewed that MSTN, PSMB5 and UCH-L3 were differentially expressed in bovine skeletal muscle satellite cells myogenic differentiation process. These data indicated ubiquitination process may play a key role in bovine muscle development. However, the role of ubiquitination process in muscle development still need well investigated. In this study, anti-ubiquitination-based enrichment and liquid chromatography-tandem mass spectrometry will be applied to comprehensively investigate the global ubiquitome in MSTN-/- cattle and wild-type cattle, select and validate the differential ubiquitinated proteins. Using bioinformatics analyses, construct interaction networks between ubiquitinated proteins and MSTN、PSMB5 and UCH-L3, analyze the possible muscle hypertrophy pathway of ubiquitination process induced by MSTN. Then the bovine skeletal muscle satellite cells induced myogenic differentiation model will be used to investigate the function and regulating mechanism of MSTN, PSMB5, UCH-L3 and their target key ubiquitinated proteins in bovine myogenic differentiation process, identify the signaling pathways of these key ubiquitinated proteins are involved, and detect the in vivo effects of the identified key ubiquitinated proteins in muscle atrophy or injury model. Through these methods, analyze the regulatory networks of ubiquitination process in bovine muscle development, and reveal the molecular mechanism of ubiquitination process in myogenic differentiation process. The results of this study could provide theoretical support for further research of ubiquitination process in bovine muscle development, and identify new target genes for the beef cattle breeding.

课题组前期通过肌肉蛋白组学分析发现MSTN-/-牛和野生型牛中两个重要的泛素蛋白水解系统的酶PSMB5和UCH-L3的表达存在显著性差异，结合在牛肌卫星细胞上的初步验证，推测泛素化途径对牛肌肉发育产生了重要的调控作用。本研究在前期研究基础上，对MSTN-/-牛和野生型牛进行肌肉泛素化蛋白组学分析，筛选泛素化修饰差异蛋白，寻找MSTN、PSMB5和UCH-L3可能调控的泛素化蛋白网络，解析MSTN通过泛素化途径调控肌肉发育的可能通路，然后利用牛骨骼肌卫星细胞成肌分化模型，对MSTN、PSMB5和UCH-L3以及它们调控的关键泛素化蛋白在牛成肌分化过程中的作用和机制进行研究，找到其参与的具体信号通路并对成肌分化关键泛素化调控蛋白进行初步的体内功能验证，综合分析泛素化途径对牛肌肉发育分化的调控作用机制，为肌肉发育分化的泛素化研究提供参考，为肉牛的分子育种研究提供有用的靶标基因。

肌肉的发育分化是一个极其复杂的过程，泛素化途径在此过程中发挥了重要的调控作用，本研究利用Label Free技术，结合LC-MS/MS技术，对MSTN-/-牛和野生型牛肌肉泛素化蛋白质组进行定量分析，从牛肌肉组织中共鉴定到135个泛素化肽段，归属于66个蛋白。与野生型牛相比，MSTN-/-牛中共鉴定到17个显著差异泛素化肽段，4个上调，13个下调，17个泛素化肽段对应了17个蛋白。生物信息学分析表明，泛素化修饰蛋白主要富集在心肌收缩、心肌病、糖酵解/糖异生以及泛素介导的蛋白水解等通路中，泛素化差异蛋白主要富集在糖酵解/糖异生、氨基酸的生物合成等通路中。STRING分析结果表明，泛素化修饰蛋白主要富集在肌肉生长发育过程、能量代谢过程和泛素蛋白水解系统中，泛素化差异蛋白主要富集在肌肉生长发育过程中。研究结果表明泛素化途径可能在MSTN介导的肌肉发育过程中发挥了重要作用，进一步将MSTN和泛素化修饰蛋白与泛素化差异蛋白进行互作分析，结果表明MSTN和肌肉生长发育过程具有密切互作关系。本研究发现MSTN、PSMB5和UCH-L3与泛素化修饰蛋白具有密切的互作关系，同时绘制出了泛素化途径对牛肌肉成肌分化过程的调控作用网络。进一步采用牛骨骼肌卫星细胞体外诱导分化模型发现MSTN、PSMB5和UCH-L3均可以调控牛骨骼肌卫星细胞的成肌分化过程。发现干扰MSTN后牛骨骼肌卫星细胞中泛素化蛋白的水平明显上调，而泛素化蛋白水平的升高对牛骨骼肌卫星细胞的成肌分化过程具有促进作用，MSTN可以通过泛素化途径影响牛的肌肉发育过程，MSTN可以通过调控泛素激活酶UBA1表达，进而调控MyoD1的表达水平，进一步调控了牛骨骼肌卫星细胞的成肌分化进程。本研究进一步构建了小鼠失神经肌损伤模型，对UBA1进行了初步的体内功能验证。本研究发现了泛素化途径在牛肌肉发育中的作用和调控机制，相关研究为牛肌肉发育分化的泛素化研究提供了理论支持，加深了对肌肉发育生长的分子遗传基础的认识，对畜禽肉用性状的遗传改良具有重要的科学意义。