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.
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