Mitsugumin 53 Research Articles

Effect of metabolic syndrome on mitsugumin 53 expression and function.

Metabolic syndrome is a cluster of risk factors, such as obesity, insulin resistance, and hyperlipidemia that increases the individual’s likelihood of developing cardiovascular diseases. Patients inflicted with metabolic disorders also suffer from tissue repair defect. Mitsugumin 53 (MG53) is a protein essential to cellular membrane repair. It facilitates the nucleation of intracellular vesicles to sites of membrane disruption to create repair patches, contributing to the regenerative capacity of skeletal and cardiac muscle tissues upon injury. Since individuals suffering from metabolic syndrome possess tissue regeneration deficiency and MG53 plays a crucial role in restoring membrane integrity, we studied MG53 activity in mice models exhibiting metabolic disorders induced by a 6 month high-fat diet (HFD) feeding. Western blotting showed that MG53 expression is not altered within the skeletal and cardiac muscles of mice with metabolic syndrome. Rather, we found that MG53 levels in blood circulation were actually reduced. This data directly contradicts findings presented by Song et. al that indict MG53 as a causative factor for metabolic syndrome (Nature 494, 375-379). The diminished MG53 serum level observed may contribute to the inadequate tissue repair aptitude exhibited by diabetic patients. Furthermore, immunohistochemical analyses reveal that skeletal muscle fibers of mice with metabolic disorders experience localization of subcellular MG53 around mitochondria. This clustering may represent an adaptive response to oxidative stress resulting from HFD feeding and may implicate MG53 as a guardian to protect damaged mitochondria. Therapeutic approaches that elevate MG53 expression in serum circulation may be a novel method to treat the degenerative tissue repair function of diabetic patients.

Tripartite motif family proteins mediate vesicular trafficking during membrane repair in striated muscle

Tripartite motif (TRIM) proteins constitute a family of proteins that all contain a canonical RING finger, B-box and coiled-coil domains which participate in several cellular processes. TRIM72, also known as mitsugumin 53 (MG53), is an essential component of the membrane repair machinery in striated muscles. The TRIM72/MG53 knockout mouse displays compromised muscle membrane repair and trim72-/- mice develop skeletal myopathy and defects following cardiovascular stress. Previous studies indicate that both endocytosis and exocytosis are important in the membrane repair process, and also that TRIM72/MG53 expression can appear in vesicles that are released from myocytes. In this study we examined the role of TRIM72/MG53 in vesicular endocytosis and exocytosis in C2C12 myoblasts overexpressing TRIM72/MG53, where overexpression of TRIM72/MG53 increased both endocytosis measured by labeled dextran entry and exocytosis as assayed by transferrin internalization and recycling. Our recent findings show that TRIM72/MG53 overexpression is sufficient to activate the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathway. Therefore, we hypothesized that this TRIM72/MG53 mediated vesicular translocation may be regulated by a PI3K-dependent pathway. To test this hypothesis, dextran uptake and transferrin internalization/recycling were measured in the presence of chemical and genetic inhibition of PI3K signaling. We conclude that TRIM72/MG53 is an important regulator of the PI3K signaling pathway and positively regulates translocation of vesicles to the site of membrane injury.

Abstract 218: The Role of Novel Tripartite Motif Proteins in Sarcolemmal Membrane Repair

Tripartite motif (TRIM) proteins are a superfamily of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly in membrane repair pathways. Mitsugumin 53 (MG53) also known as TRIM72, is primary expressed in skeletal muscle and heart. Our experimental data confirm that during membrane damage, MG53 translocates to the injury site and acts as a molecular glue to reseal the damage area. The role of MG53 in membrane repair has been demonstrated in both in vitro studies using molecular approaches and in vivo using rodent wild type and knockout models. Thus, our data indicate that recombinant human MG53 protein can be directly applied as a therapeutic agent to increase the membrane repair capacity of many cell types, including cardiomyocytes during acute injury or in chronic disease progression. However, the precise mechanism and potential partners by which MG53 executes its membrane repair function are not completely understood. On the basis of the global TRIM family protein alignment, we hypothesize that there are other TRIM proteins that, alone or together with MG53, may facilitate repair by targeting the site of an injury. Moreover, data from our lab demonstrated that MG53 and these TRIM proteins can form homo- and hetero-oligomeric assemblies due to the presence of the coiled-coil region in these proteins and, further, that this may be necessary for the active membrane resealing process. Using E. coli protein expression methodology we can generate and isolate new TRIM recombinant proteins and test if these protein complexes are effective when applied externally to cardiac and non-cardiac cells. These novel proteins will also be tested for their pharmacokinetic properties to determine their efficacy in both acute and chronic applications. Our studies should increase our knowledge of the mechanisms controlling cardiac membrane repair and also provide novel therapeutic targets.

Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.

Injury to lung epithelial cells has a role in multiple lung diseases. We previously identified mitsugumin 53 (MG53) as a component of the cell membrane repair machinery in striated muscle cells. Here we show that MG53 also has a physiological role in the lung and may be used as a treatment in animal models of acute lung injury. Mice lacking MG53 show increased susceptibility to ischemia-reperfusion and over-ventilation induced injury to the lung when compared with wild type mice. Extracellular application of recombinant human MG53 (rhMG53) protein protects cultured lung epithelial cells against anoxia/reoxygenation-induced injuries. Intravenous delivery or inhalation of rhMG53 reduces symptoms in rodent models of acute lung injury and emphysema. Repetitive administration of rhMG53 improves pulmonary structure associated with chronic lung injury in mice. Our data indicate a physiological function for MG53 in the lung and suggest that targeting membrane repair may be an effective means for treatment or prevention of lung diseases.

MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis and insulin signalling

Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is an ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme, UBE2H. Molecular manipulations that disrupt the E3-ligase function of MG53 abolish IRS-1 ubiquitination and enhance skeletal myogenesis. Skeletal muscles derived from the MG53-/- mice show an elevated IRS-1 level with enhanced insulin signalling, which protects the MG53-/- mice from developing insulin resistance when challenged with a high-fat/high-sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.

TRIM Proteins in Therapeutic Membrane Repair of Muscular Dystrophy

Muscular dystrophy represents a major unmet medical need; only palliative treatments exist for this group of debilitating diseases. Because multiple forms of muscular dystrophy arise from compromised sarcolemmal membrane integrity, a therapeutic approach that can target this loss of membrane function could be applicable to a number of these distinct diseases.One promising therapeutic approach involves the process the cell uses to repair injuries to the plasma membrane. Recent discoveries of genes associated with the membrane repair process provide an opportunity to promote this process as a way to treat muscular dystrophy. One such gene is mitsugumin 53 (MG53), a member of the tripartite motif (TRIM) family of proteins (TRIM72), which is an essential component of the membrane repair pathway in muscle. Recent results indicate that MG53/TRIM72 protein can be directly applied as a therapeutic agent to increase membrane repair capacity of many cell types and treat some aspects of the disease in mouse models of muscular dystrophy. There is great potential for the use of recombinant human MG53 in treating muscular dystrophy and other diseases in which compromised membrane integrity contributes to the disease. Other TRIM family proteins may provide additional targets for therapeutic intervention in similar disease states.

The role of mitsugumin 53 in cell membrane repair and myocardial protection

Background Mitsugumin 53(MG53),specially expressed in skeletal and cardiac muscle,is a newly discovered member of Tripartite Motif Family Proteins.Objective Explore the molecular mechanisms of the MG53 involved in the muscle membrane repair and myocardial protection.Content Discuss the role of MG53 in following details:the molecular mechanisms of the MG53 involved in the muscle membrane repair and the role in myocardial ischemic preconditioning and postconditioning protection mechanisms.Trend The value of newly discovered MG53 in myocardial protection needs further study,it will provide new methods for the diagnosis and treatment in heart disease. Key words: Mitsugumin 53 ; Cell membrane repair ; Myocardial protection ;

Calpains, Cleaved Mini-DysferlinC72, and L-Type Channels Underpin Calcium-Dependent Muscle Membrane Repair

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

Mitsugumin 53 Attenuates the Activity of Sarcoplasmic Reticulum Ca2+-ATPase 1a (SERCA1a) in Skeletal Muscle

Mitsugumin 53 (MG53) is a member of membrane repair system in skeletal muscle. However, role(s) of MG53 in unique functions of skeletal muscle has not been addressed although MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were searched among proteins mediating skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knock-down in mouse primary skeletal myotubes increased Ca2+-uptake through SERCA1a (more than 35%) at micromolar Ca2+ but not at nanomolar Ca2+, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. In-silico studies suggest that the binding of MG53 to SERCA1a is mediated by unique ways compared with bindings by other proteins containing TRIM or PRY domains.

Mitsugumin 53 protects the kidney from severe burn injury in mice.

Mitsugumin 53 (MG53), a newly identified muscle-specific protein, is an essential component of the cell membrane repair machinery in skeletal and cardiac muscle. However, the role of MG53 after burns in other tissues remains unclear. This study aims to investigate the possible roles of MG53 in the protection of the kidney after severe burn injury, and an animal scalding model of 30% of total body surface area (TBSA) was used. Recombinant human MG53 (rhMG53) or bovine serum albumin (BSA) was injected intravenously via the tail vein. Data showed that the mortality in the MG53-treated group was lower than that in control group. Administration of rhMG53 may alleviate histological alterations in renal tubular epithelial cells after burn injury. Renal tubular injury scores and the average optical density score of kidney injury molecule-1 (KIM-1) immunohistochemical staining in the MG53-treated group were significantly lower than those in control group (P < 0.001). Exogenous rhMG53 was found to be located in renal tubular epithelial cells. Numerous polymerase I and transcript release factor (PTRF) were expressed in the mouse kidney after severe scalding. In conclusion, our data indicate that MG53 protein protects the kidney by involving local PTRF after severe burn injury.

Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca2+-uptake through SERCA1a (more than 35%) at micromolar Ca2+ but not at nanomolar Ca2+, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.

The C2A domain in dysferlin is important for association with MG53 (TRIM72)

In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild-type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP-tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair.

Dysferlin, Annexin A1, and Mitsugumin 53 Are Upregulated in Muscular Dystrophy and Localize to Longitudinal Tubules of the T-System With Stretch

Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

Muscle membrane repair and inflammatory attack in dysferlinopathy

Repair of plasma membrane tears is an important normal physiological process that enables the cells to survive a variety of physiological and pathological membrane lesions. Dysferlin was the first protein reported to play a crucial role in this repair process in muscle, and recently, several other proteins including Mitsugumin 53 (MG53), annexin and calpain were also found to participate. These findings have now established the framework of the membrane repair mechanism. Defective membrane repair in dysferlin-deficient muscle leads to the development of muscular dystrophy associated with remarkable muscle inflammation. Recent studies have demonstrated a crosstalk between defective membrane repair and immunological attack, thus unveiling a new pathophysiological mechanism of dysferlinopathy. Here I summarize and discuss the latest progress in the molecular mechanisms of membrane repair and the pathogenesis of dysferlinopathy. Discussion about potential therapeutic applications of these findings is also provided.

Membrane Repair Defects in Muscular Dystrophy Are Linked to Altered Interaction between MG53, Caveolin-3, and Dysferlin

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.

Mitsugumin 53 (MG53) facilitates vesicle trafficking in striated muscle to contribute to cell membrane repair

Repair of the plasma membrane following damage is an important aspect of normal cellular physiology, and disruption of this process is observed in many pathologic states. In a recent series of publications, we resolved that Mitsugumin 53 (MG53) is a novel, muscle-specific member of the tripartite motif/RING B-box Coiled Coil (TRIM/RBCC) family of proteins (TRIM72) that contributes to vesicle trafficking in the course of normal cellular physiology. MG53 can bind phosphatidylserine (PS) with some specificity, and interacts with caveolin-3 (Cav-3) as part of its function in vesicle trafficking. As part of the response to membrane damage in muscle cells, MG53 acts in an oxidation-dependent manner to facilitate vesicle translocation to the sites of membrane injury where these vesicles are involved in patching the membrane. Here we discuss these findings and examine the implications of this work in the field of membrane repair. Further discussion is provided about potential therapeutic applications for MG53.Addendum to: Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M, Ko JK, Lin P, Thornton A, Zhao X, Pan Z, Komazaki S, Brotto M, Takeshima H, Ma J. MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol 2009; 11:56–64; PMID: 19043407; DOI: 10.1038/ncb1812. Cai C, Masumiya H, Weisleder N, Pan Z, Nishi M, Komazaki S, Takeshima H, Ma J. MG53 regulates membrane budding and exocytosis in muscle cells. J Biol Chem 2009; 284:3314–22; PMID: 19029292; doi:10.1074/jbc.M808866200.andMasumiya H, Asaumi Y, Nishi M, Minamisawa S, Adachi-Akahane S, Yoshida M, Kangawa K, Ito K, Kagaya Y, Yanagisawa T, Yamazaki T, Ma J, Takeshima H. Mitsugumin 53-mediated maintenance of K+ currents in cardiac myocytes. Channels 2009; In press.

Mitsugumin 53-mediated maintenance of K+ currents in cardiac myocytes

Mitsugumin 53 (MG53) is a muscle-specific RBCC/TRIM family member predominantly localized on small vesicles underneath the plasma membrane. Upon cell-surface lesion MG53 recruits the vesicles to the repair site in an oxidation-dependent manner and MG53-knockout mice develop progressive myopathy associated with defective membrane repair. In this report, we focus on MG53-knockout cardiomyocytes showing abnormal action potential profile and a reduced K+ current density. In cDNA expression experiments using cultured cells, KV2.1-mediated currents were remarkably increased by MG53 without affecting the total and cell-surface levels of channel expression. In imaging analysis MG53 seemed to facilitate the mobility of KV2.1-containing endocytic vesicles with acidic pH. However, similar effects on the current density and vesicular mobility were not observed in the putative dominant-negative form of MG53. Our data suggest that MG53 is involved in a constitutive cycle of certain cell-surface proteins between the plasma membrane and endosome-like vesicles in striated muscle, and also imply that the vesicular dynamics are essential for the quality control of KV2.1 in cardiomyocytes.