Edited by Jeffrey PessinMitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance.Noninsulin-dependent diabetes mellitus (type 2 diabetes) has become a worldwide epidemic disease due to the increased incidence of obesity. Insulin receptor (IR) and insulin receptor substrate (IRS) 4 are inactivated by elevated serum levels of free fatty acids, leading to insulin resistance and noninsulin-dependent diabetes mellitus (1-3). Because skeletal muscle is the largest organ participating in glucose uptake, exercise-induced skeletal muscle development is an excellent treatment for insulin resistance. However, no current candidates target skeletal muscle to treat insulin resistance.Mitsugumin 53 (MG53), which is also called tripartite motifcontaining protein 72 (TRIM72) and which is largely expressed in skeletal muscle, was independently identified by proteomic analysis of lipid rafts and triad-rich membranes (4). MG53 contains a tripartite domain (an E3 ligase RING domain, a B-box, and two coiled-coil domains) and a SPRY domain. Insulin-like growth factor-1 (IGF-1) initiates MyoD activation via an IGF-1 receptor-PI3K-Akt pathway during skeletal myogenesis (5, 6). MyoD and myocyte enhancer factor 2 (MEF2) binds to two proximal E-boxes and an MEF2 site in MG53 promoter, activating MG53 gene transcription (7). The MG53 protein interacts with IRS-1 and focal adhesion kinase (FAK), inducing IRS-1 and FAK ubiquitination and degradation in skeletal muscle with the help of E2 ligase UBE2H (8 -10). Moreover, RING domain-disrupted MG53 mutants (⌬R and C14A) abolish IRS-1 and FAK ubiquitination and degradation in skeletal muscle, indicating that MG53 is an E3 ligase that targets IRS-1 and FAK.Systemic MG53 ablation abrogates IRS-1 ubiquitination and degradation in skeletal and cardiac muscle, leading to elevated IRS-1 expression level and increased insulin signaling (8, 9). Thus, MG53 knock-out mice do not develop diet-induced insulin resistance. In contrast, skeletal muscle-specific MG53 transgenic mice exhibit metabolic disorders such as obesity, ...

Although proteomic analyses have revealed the presence of mitochondrial oxidative phosphorylation (OXPHOS) proteins in the plasma membrane, there have been no in-depth evaluations of the presence or function of OXPHOS I-V in the plasma membrane. Here, we demonstrate the in situ localization of OXPHOS I-V complexes to the sarcolemma of skeletal muscle by immunofluorescence and immunohistochemistry. A portion of the OXPHOS I-V complex proteins was not co-stained with MitoTracker but co-localized with caveolin-3 in the sarcolemma of mouse gastrocnemius. Mitochondrial matrix-facing OXPHOS complex subunits were ectopically expressed in the sarcolemma of the non-permeabilized muscle fibers and C2C12 myotubes. The sarcolemmal localization of cytochrome c was also observed from mouse gastrocnemius muscles and C2C12 myotubes, as determined by confocal and total internal resonance fluorescence (TIRF) microscopy. Based on these data, we conclude that a portion of OXPHOS complexes is localized in the sarcolemma of skeletal muscle and may have non-canonical functions. [BMB Reports 2016; 49(2): 116-121]