Specificity and Activity of Ozz-E3 During Myogenesis

描述(由申请人提供)：调控肌肉发生的泛素蛋白连接酶的身份在很大程度上是未知的。然而，我们已经确定了一个编码一种新的细胞因子信号转导抑制蛋白077的基因，该蛋白仅在横纹骨骼肌和心肌中表达。OZZ与Elongin B/C复合体相互作用，进而促进环型泛素连接酶的组装，其底物可能是a-连环蛋白和胚胎肌球蛋白重链(MyHCemb)。OZZ的表达在肌肉发生过程中受到调节；在成肌细胞中，蛋白酶体迅速降解OZZ的磷酸化形式，而在肌管中，SoCS盒中酪氨酸残基的去磷酸化稳定了OZZ，并使其能够结合OZZ-E3连接酶复合体的其他成分。我们的假设是，Ozz-E3连接酶复合体在成人MyHC发育异构体的交换以及肌节组装成由a-catenin稳定的肌原纤维中发挥关键作用；这两个过程都允许肌肉纤维的分化和重塑。这项建议的目的是通过研究OZZ蛋白表达的调节机制、077在肌肉发生过程中的功能以及OZZ调节其指定的E3连接酶复合体的活性的方式来检验这一假说。 在第一个特定目标中，我们将研究在体外肌肉细胞分化过程中内源性Ozz-E3连接酶复合体的组装和调控。在肌肉细胞分化的不同阶段，我们将使用多种分子和生化方法来纯化内源性Ozz-E3连接酶复合体，并分析Ozz-E3连接酶的泛素化活性。在第二个具体目标中，我们将在体内研究Ozz在肌肉发生和肌肉动态平衡中的作用。我们已经产生了一种在Ozz基因座携带零突变的小鼠品系。这些小鼠将有助于详细分析臭氧介导的泛素化在肌肉发生过程中的功能。我们将特别关注Ozz-E3复合体在横纹肌发育、生长和老化过程中肌节重塑和成熟过程中的潜在作用。了解OZZ-E3连接酶复合体的作用将为控制肌肉分化和再生早期阶段的机制提供新的见解；阐明这些调节机制也可能为心脏和骨骼肌病的治疗提供新的见解。

DESCRIPTION (provided by applicant): The identities of the ubiquitin-protein ligases that regulate myogenesis are largely unknown. However, we have identified a gene that encodes a novel suppressor of cytokine signaling (SOCS) protein, 077, which is expressed exclusively in striated skeletal and cardiac muscle. Ozz interacts with the Elongin B/C complex and, in turn, promotes the assembly of a RING-type ubiquitin ligase whose putative substrates are a-catenin and embryonic myosin heavy chain (MyHCemb). The expression of Ozz is regulated during myogenesis; in myoblasts, the proteasome rapidly degrades the phosphorylated form of Ozz, and in myotubes, the dephosphorylation of a tyrosine residue within the SOCS box stabilizes Ozz and enables it to bind the other components of the Ozz-E3 ligase complex. Our hypothesis is that the Ozz-E3 ligase complex plays a crucial role in the regulation of the exchange of developmental isoforms of MyHC for the adult forms and in the assembly of sarcomeres into myofibrils stabilized by a-catenin; both processes permit differentiation and remodeling of muscle fibers. The objective of this proposal is to test this hypothesis by investigating the mechanisms that regulate the expression of Ozz protein, the function of 077 during myogenesis, and the manner in which Ozz regulates the activity of the E3 ligase complex that it specifies. In the first specific aim, we will characterize the assembly and regulation of the endogenous Ozz-E3 ligase complex during the differentiation of muscle cells in vitro. Several molecular and biochemical approaches will be used to purify the endogenous Ozz-E3 ligase complex at different stages of muscle-cell differentiation and to analyze the ubiquitination activity of Ozz-E3 ligase. In the second specific aim, we will study in vivo the function of Ozz in myogenesis and muscle homeostasis. We have generated a mouse strain carrying a null mutation at the Ozz locus. These mice will facilitate the detailed analysis of the function of Ozz-mediated ubiquitination during myogenesis. We will focus specifically on the potential role of the Ozz-E3 complex in the regulation of sarcomere remodeling and maturation during development, growth, and aging of striated muscle. Understanding the role of the Ozz-E3 ligase complex will provide new insight about the mechanisms that control the early stages of muscle differentiation and regeneration; elucidation of these regulatory mechanisms may also provide new insight regarding treatment for cardiac and skeletal myopathies.