Tao Tan

Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy

Recombinant human MG53 protein can increase membrane repair after injury in cells and can reduce pathology in animal models of muscle injury and muscular dystrophy. Mending Muscle To repair a torn muscle, one might require a little bit of ice and a lot of rest. For those with Duchenne muscular dystrophy (DMD), however, muscle degeneration is not as easily repaired, and patients ultimately experience difficulty standing, walking, and breathing. DMD results from a lack of the protein dystrophin, which is located at the cell membrane to help muscle fibers repair themselves. There is no cure for DMD, but Weisleder and colleagues have now shown that exogenous delivery of a different repair protein, Mitsugumin 53 (MG53), to cells can prevent muscle damage in cell culture and in mice. The authors first showed that muscle and nonmuscle cells treated with recombinant human MG53 (rhMG53) in vitro were resistant to mechanical, chemical, and photo damage because MG53 localized to the injury site and provided protection. In vivo, Weisleder and colleagues showed that dystrophin-deficient mdx mice treated intramuscularly or intravenously with rhMG53 displayed reduced muscle damage and decreased muscle pathology compared to saline-treated controls, even in the presence of a membrane-damaging toxin. This repair process also worked in muscle fibers isolated from mdx mice that were deficient in either of two natural repair proteins, MG53 or dysferlin, suggesting that exogenous delivery of rhMG53 works by a new mechanism—other than the intracellular machinery—to patch up damaged cell membrane. Soluble MG53 protein therapy could be a viable treatment for DMD that avoids the well-known limitations of dystrophin gene replacement therapy. Toward translation, Weisleder et al. have further demonstrated that exogenous MG53 is nontoxic and safe in animals. The ability of the protein to preserve muscle function and to enhance repair capacity in humans has yet to be shown, but additional studies in larger animals and human muscle fibers will give a clearer indication of its therapeutic potential. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein, is an essential component of the cell membrane repair machinery. Here, we examined the translational value of targeting MG53 function in tissue repair and regenerative medicine. Although native MG53 protein is principally restricted to skeletal and cardiac muscle tissues, beneficial effects that protect against cellular injuries are present in nonmuscle cells with overexpression of MG53. In addition to the intracellular action of MG53, injury to the cell membrane exposes a signal that can be detected by MG53, allowing recombinant MG53 protein to repair membrane damage when provided in the extracellular space. Recombinant human MG53 (rhMG53) protein purified from Escherichia coli fermentation provided dose-dependent protection against chemical, mechanical, or ultraviolet-induced damage to both muscle and nonmuscle cells. Injection of rhMG53 through multiple routes decreased muscle pathology in the mdx dystrophic mouse model. Our data support the concept of targeted cell membrane repair in regenerative medicine, and present MG53 protein as an attractive biological reagent for restoration of membrane repair defects in human 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.

Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury

Ischemic heart disease is a leading cause of death in human population and protection of myocardial infarction (MI) associated with ischemia-reperfusion (I/R) remains a challenge. MG53 is an essential component of the cell membrane repair machinery that protects injury to the myocardium. We investigated the therapeutic value of using the recombinant human MG53 (rhMG53) protein for treatment of MI. Using Langendorff perfusion of isolated mouse heart, we found that I/R caused injury to cardiomyocytes and release of endogenous MG53 into the extracellular solution. rhMG53 protein was applied to the perfusion solution concentrated at injury sites on cardiomyocytes to facilitate cardioprotection. With rodent models of I/R-induced MI, we established the in vivo dosing range for rhMG53 in cardioprotection. Using a porcine model of angioplasty-induced MI, the cardioprotective effect of rhMG53 was evaluated. Intravenous administration of rhMG53, either prior to or post-ischemia, reduced infarct size and troponin I release in the porcine model when examined at 24h post-reperfusion. Echocardiogram and histological analyses revealed that the protective effects of rhMG53 observed following acute MI led to long-term improvement in cardiac structure and function in the porcine model when examined at 4weeks post-operation. Our study supports the concept that rhMG53 could have potential therapeutic value for treatment of MI in human patients with ischemic heart diseases.

MG53-mediated cell membrane repair protects against acute kidney injury

Recombinant MG53 translocates to sites of injury in the proximal tubule of the kidney and protects mice from acute kidney injury induced by ischemia or drugs. A molecular bandage for kidney injury MG53 is a protein that is primarily expressed in muscles and helps protect muscle cells from damage. Now, Duann et al. have shown that MG53 performs a similar function in the kidney as well. The authors evaluated the role of MG53 in mouse models of kidney injury induced by ischemia and reperfusion, as well as by cisplatin, a highly nephrotoxic chemotherapy drug. In each case, recombinant MG53 could be given intravenously, and the authors found that it bound to the sites of injury on kidney cells and protected them from further damage and death. MG53 treatment did not interfere with the effectiveness of cisplatin against cancer cells, suggesting that MG53 may be useful for protecting patients’ kidneys during chemotherapy. Injury to the renal proximal tubular epithelium (PTE) represents the underlying consequence of acute kidney injury (AKI) after exposure to various stressors, including nephrotoxins and ischemia/reperfusion (I/R). Although the kidney has the ability to repair itself after mild injury, insufficient repair of PTE cells may trigger inflammatory and fibrotic responses, leading to chronic renal failure. We report that MG53, a member of the TRIM family of proteins, participates in repair of injured PTE cells and protects against the development of AKI. We show that MG53 translocates to acute injury sites on PTE cells and forms a repair patch. Ablation of MG53 leads to defective membrane repair. MG53-deficient mice develop pronounced tubulointerstitial injury and increased susceptibility to I/R-induced AKI compared to wild-type mice. Recombinant human MG53 (rhMG53) protein can target injury sites on PTE cells to facilitate repair after I/R injury or nephrotoxin exposure. Moreover, in animal studies, intravenous delivery of rhMG53 ameliorates cisplatin-induced AKI without affecting the tumor suppressor efficacy of cisplatin. These findings identify MG53 as a vital component of reno-protection, and targeting MG53-mediated repair of PTE cells represents a potential approach to prevention and treatment of AKI.

Delivery of placenta-derived mesenchymal stem cells ameliorates ischemia induced limb injury by immunomodulation.

Background: Peripheral artery disease (PAD) is a major health burden in the world. Stem cell-based therapy has emerged as an attractive treatment option in regenerative medicine. In this study, we sought to test the hypothesis that stem cell-based therapy can ameliorate ischemia induced limb injury. Methods: We isolated mesenchymal stem cells derived from human placentas (PMSCs) and intramuscularly transplanted them into injured hind limbs. Treatment with PMSCs reduced acute muscle fibers apoptosis induced by ischemia. Results: PMSC treatment significantly enhanced regeneration of the injured hind limb by reducing fibrosis and enhancing running capacity when the animals were subjected to treadmill training. Mechanistically, injected PMSCs can modulate acute inflammatory responses by reducing neutrophil and macrophage infiltration following limb ischemia. ELISA assays further confirmed that PMSC treatment can also reduce pro-inflammatory cytokines, TNF-α and IL-6, and enhance anti-inflammatory cytokine, IL-10 at the injury sites. Conclusion: Taken together, our results demonstrated that PMSCs can be a potential effective therapy for treatment of PAD via immunomodulation.

Dual function of MG53 in membrane repair and insulin signaling

MG53 is a member of the TRIM-family protein that acts as a key component of the cell membrane repair machinery. MG53 is also an E3-ligase that ubiquinates insulin receptor substrate-1 and controls insulin signaling in skeletal muscle cells. Since its discovery in 2009, research efforts have been devoted to translate this basic discovery into clinical applications in human degenerative and metabolic diseases. This review article highlights the dual function of MG53 in cell membrane repair and insulin signaling, the mechanism that underlies the control of MG53 function, and the therapeutic value of targeting MG53 function in regenerative medicine. [BMB Reports 2016; 49(8): 414-423]

Amelioration of ischemia‐reperfusion–induced muscle injury by the recombinant human MG53 protein

Introduction: Ischemia‐reperfusion injury (I‐R) in skeletal muscle requires timely treatment. Methods: Rodent models of I‐R injury were used to test the efficacy of recombinant human MG53 (rhMG53) protein for protecting skeletal muscle. Results: In a mouse I‐R injury model, we found that mg53,−/− mice are more susceptible to I‐R injury. rhMG53 applied intravenously to the wild‐type mice protected I‐R injured muscle, as demonstrated by reduced CK release and Evans blue staining. Histochemical studies confirmed beneficial effects of rhMG53. Of interest, rhMG53 did not protect against I‐R injury in rat skeletal muscle. This was likely due to the fact that the plasma level of endogenous MG53 protein is high in rats. Conclusions: Our data suggest that rhMG53 may be a potential therapy for protection against muscle trauma. A mouse model appears to be a better choice than a rat model for evaluating potential treatments for protecting skeletal muscle. Muscle Nerve 52: 852–858, 2015

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes

Background: MG53, a zinc finger protein, is essential to cell membrane repair. It is not known whether zinc contributes to MG53-mediated membrane repair. Results: Chelation of Zn2+ or mutation of Zn2+-binding motifs in MG53 affects membrane repair. Conclusion: Zn2+ binding to MG53 is required for membrane repair. Significance: This study establishes a base for Zn2+ interaction with MG53 in protection against injury to the cell membrane. Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn2+ deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn2+-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn2+-binding motifs. Here, we show that Zn2+ binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn2+ entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn2+ on membrane repair is abolished in mg53−/− muscle fibers, suggesting that MG53 functions as a potential target for Zn2+ during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn2+-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn2+ interaction with MG53 in protection against injury to the cell membrane.

MG53 permeates through blood-brain barrier to protect ischemic brain injury

Ischemic injury to neurons represents the underlying cause of stroke to the brain. Our previous studies identified MG53 as an essential component of the cell membrane repair machinery. Here we show that the recombinant human (rh)MG53 protein facilitates repair of ischemia-reperfusion (IR) injury to the brain. MG53 rapidly moves to acute injury sites on neuronal cells to form a membrane repair patch. IR-induced brain injury increases permeability of the blood-brain-barrier, providing access of MG53 from blood circulation to target the injured brain tissues. Exogenous rhMG53 protein can protect cultured neurons against hypoxia/reoxygenation-induced damages. Transgenic mice with increased levels of MG53 in the bloodstream are resistant to IR-induced brain injury. Intravenous administration of rhMG53, either prior to or after ischemia, can effectively alleviate brain injuries in rats. rhMG53-mediated neuroprotection involves suppression of apoptotic neuronal cell death, as well as activation of the pro-survival RISK signaling pathway. Our data indicate a physiological function for MG53 in the brain and suggest that targeting membrane repair or RISK signaling may be an effective means to treat ischemic brain injury.

DEPTOR suppresses the progression of esophageal squamous cell carcinoma and predicts poor prognosis.

As a naturally occurring inhibitor of mTOR, accumulated evidence has suggested that DEPTOR plays a pivotal role in suppressing the progression of human malignances. However, the function of DEPTOR in the development of esophageal squamous cell carcinoma (ESCC) is still unclear. Here we report that the expression of DEPTOR is significantly reduced in tumor tissues derived from human patients with ESCC, and the downregulation of DEPTOR predicts a poor prognosis of ESCC patients. In addition, we found that the expression of DEPTOR negatively regulates the tumorigenic activities of ESCC cell lines (KYSE150, KYSE510 and KYSE190). Furthermore, ectopic DEPTOR expression caused a significant suppression of the cellular proliferation, migration and invasion of KYSE150 cells, which has the lowest expression level of DEPTOR in the three cell lines. Meanwhile, CRISPR/Cas9 mediated knockout of DEPTOR in KYSE-510 cells significantly promoted cellular proliferation, migration and invasion. In addition, in vivo assays further revealed that tumor growth was significantly inhibited in xenografts with ectopic DEPTOR expression as compared to untreated KYSE150 cells, and was markedly enhanced in DEPTOR knockout KYSE-510 cells. Biochemical studies revealed that overexpression of DEPTOR led to the suppression of AKT/mTOR pathway as evidenced by reduced phosphorylation of AKT, mTOR and downstream SGK1, indicating DEPTOR might control the progression of ESCC through AKT/mTOR signaling pathway. Thus, these findings, for the first time, demonstrated that DEPTOR inhibits the tumorigenesis of ESCC cells and might serve as a potential therapeutic target or prognostic marker for human patients with ESCC.