Specificity and Activity of Ozz-E3 During Myogenesis

Ozz-E3 在肌生成过程中的特异性和活性

• 批准号: 6891695
• 负责人: ALESSANDRA D'AZZO
• 金额: $ 23.69万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-02 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891695
• 关键词: biological signal transduction; cytokine; enzyme complex; genetically modified animals; laboratory mouse; ligase; molecular assembly /self assembly; myogenesis; myosins; protein structure function; sarcomeres; striated muscles; ubiquitin

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.

描述（由申请人提供）：调节肌生成的泛素-蛋白质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基因，编码一种新的抑制细胞因子信号（SOCS）蛋白，077，这是专门在横纹骨骼肌和心肌表达。Ozz与延伸蛋白B/C复合物相互作用，进而促进RING型泛素连接酶的组装，该酶的推定底物是α-连环蛋白和胚胎肌球蛋白重链（MyHCem B）。Ozz的表达在肌生成过程中受到调节;在成肌细胞中，蛋白酶体快速降解Ozz的磷酸化形式，在肌管中，SOCS盒内酪氨酸残基的去磷酸化使Ozz稳定，并使其能够结合Ozz-E3连接酶复合物的其他组分。我们的假设是，Ozz-E3连接酶复合物在调节成人形式的MyHC发育亚型的交换和在肌节组装成由α-连环蛋白稳定的肌原纤维中起着至关重要的作用;这两个过程都允许肌纤维的分化和重塑。本提案的目的是通过研究Ozz蛋白表达的调节机制、肌生成过程中077的功能以及Ozz调节其指定的E3连接酶复合物活性的方式来验证这一假设。 在第一个具体的目标，我们将在体外肌肉细胞分化过程中的内源性Ozz-E3连接酶复合物的组装和调节的特点。几种分子和生物化学方法将被用来纯化的内源性Ozz-E3连接酶复合物在不同阶段的肌肉细胞分化和分析的泛素化活性的Ozz-E3连接酶。在第二个具体目标中，我们将在体内研究Ozz在肌肉发生和肌肉稳态中的功能。我们已经产生了在Ozz基因座处携带无效突变的小鼠品系。这些小鼠将有助于详细分析Ozz-mediated泛素化在肌发生过程中的功能。我们将特别关注Ozz-E3复合物在横纹肌发育、生长和衰老过程中肌节重塑和成熟调节中的潜在作用。了解Ozz-E3连接酶复合物的作用将为控制肌肉分化和再生的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变的治疗提供新的见解。