Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca 2+ facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca 2+ -dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.To maintain cellular homeostasis, eukaryotic cells must conserve the integrity of their plasma membrane through active recycling and repair in response to various sources of damage 1 . Defects in the intrinsic membrane repair response have been linked to numerous disease states, including muscular dystrophy, heart failure and neurodegeneration [2][3][4][5] . Repair of plasma membrane damage requires recruitment of intracellular vesicles to injury sites 6,7 . One protein that has been linked to membrane repair in skeletal muscle is dysferlin [8][9][10] , which is thought to act as a fusogen that participates in restoration of sarcolemmal membrane integrity following muscle injury. Evidence for this role of dysferlin comes, in part, from studies showing that ablation of dysferlin in mice results in muscular dystrophy 8 .Repair of damage to the plasma membrane is an active and dynamic process that requires several steps, including participation of molecular sensor(s) that can detect acute injury to 6 Correspondence should be addressed to J.M. or H.T. (maj2@umdnj.edu; takeshim@pharm.kyoto-u.ac.jp).Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 November 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the plasma membrane, nucleation of intracellular vesicles at the injury site and vesicle fusion to enable membrane patch formation. It is well demonstrated that entry of extracellular Ca 2+ is involved in the fusion of intracellular vesicles to reseal the injured plasma membrane 6,11,12 , whereas the molecular machinery involved in sensing the damaged membrane signal and the nucleation process for repair-patch formation have not been fully resolved.We have previously established an immunopr...

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.

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.Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis and maintenance of sarcolemma integrity in striated muscle. During the life cycle of striated muscle, membrane repair is a fundamental process in maintaining cellular integrity, as shown by recent studies that link defective membrane repair to the progression of muscular dystrophy (1-3). Repair of the plasma membrane damage requires recruitment of intracellular vesicles to injury sites (4, 5). One protein that has been linked to membrane repair in skeletal muscle is dysferlin (6, 7), which is thought to fuse intracellular vesicles to patch the damaged membrane and restore sarcolemmal integrity following muscle injury. Like dysferlin, caveolin-3 (Cav3) 3 is a muscle-specific protein, and many mutations in Cav3, including P104L, R26Q, and C71W, have been linked to muscular dystrophy (8 -11). Despite extensive research efforts on Cav3 and dysferlin (12)(13)(14), the molecular function of these two proteins in membrane repair in muscle physiology and dystrophy have not been fully defined.Animal model studies reveal that either loss or gain of Cav3 function both result in dystrophic phenotypes in skeletal muscle (15, 16), suggesting that associated cellular components may be involved in the etiology of Cav3-related dystrophy. Although the discovery of dysferlin highlights the importance of membrane repair in the etiology of muscular dystrophy, dysferlin itself does not appear to participate in recruitment of intracellular vesicles because dysferlin Ϫ/Ϫ muscle retains accumulation of vesicles near membrane damage sites (7). This indicates that proteins other than dys...

Ca 2þ -triggered membrane fusion, the defining step of exocytosis, enables temporal/spatial control over the release of biologically active compounds. The mechanism by which Ca 2þ triggers and modulates native membrane fusion is still poorly understood. As an unbiased approach to investigating this process, the effects of several thiol-reactive reagents on the homotypic fusion of isolated cortical vesicles (a stage-specific preparation for analyses of native Ca 2þ -triggered fusion) have been characterized. Such reagents have been consistently shown to inhibit the Ca 2þ -sensitivity, rate and extent of triggered fusion. However, we recently showed that iodoacetamide can also potentiate the Ca 2þ -sensitivity and rate of release [1]. This implicates two distinct thiol sites in the fusion process -one involved in the ability of vesicles to fuse (extent) and one that modulates fusion efficiency (Ca 2þ -sensitivity and kinetics). Capitalizing on this potentiating effect, we have now identified other fluorescent thiol-reactive reagents with similar effects: treatment with Lucifer yellow iodoacetamide, monobromobimane or dibromobimane resulted in an average leftward shift in EC 50 from 17.251.6mM to 8.951.9mM [Ca 2þ ] free . These fluorescent reagents can be used to enhance fusion and label proteins involved in the Ca 2þ -sensing mechanism. The lipid matrix at or near the fusion site can also modulate the fusion process, specifically via cholesterol-and sphingomyelin-enrichment that is thought to regulate the Ca 2þsensitivity and rate of fusion through spatial organization of critical lipids and proteins [2,3]. Proteins involved in Ca 2þ -sensing are thus likely to be situated within such areas of the membrane. Isolation of fluorescently labeled proteins from cholesterol-enriched vesicle membrane fractions by 2-dimesional electrophoresis is now being used to identify proteins potentially involved in the Ca 2þ -triggering steps of membrane fusion.

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.When myoblasts exit the cell cycle during myogenesis, dramatic changes in membrane organization occur as myoblast fusion allows the formation of multinucleated muscle fibers. In addition to cell fusion events, differentiation of myotubes involves establishment of specialized membrane structures (1, 2). The transverse tubular invagination of sarcolemmal membrane and the intracellular membrane network known as the sarcoplasmic reticulum are two highly organized membrane architectures in cardiac and skeletal muscle. Establishment of these intricate membrane compartments requires extensive remodeling of the immature myoblast membranes. Dynamic membrane remodeling also contributes to many physiologic processes in mature muscle, including Ca 2ϩ signaling, trafficking of glucose transporter (GLUT4), and other membrane internalization events involving caveolae structures (3-6). Although defects in membrane integrity have been linked to various forms of muscular dystrophy (7,8), the molecular machinery regulating these specific membrane recycling and remodeling events in striated muscle is not well defined.The large tripartite motif (TRIM) 5 family of proteins is involved in numerous cellular functions in a wide variety of cell types. Members of this protein family contain signature motifs that include a RING finger, a zinc binding moiety (B-box), and a coiled coil structure (RBCC), which invariably comprise the amino-terminal domain of TRIM family members (9). The carboxyl-terminal sequence of TRIM proteins is variable; in some cases a subfamily of TRIM proteins contains a SPRY domain, a sequence first observed in the ryanodine receptor Ca 2ϩ channel in the sarcoplasmic reticulum membrane of excitable cells (10). Extensive studies have revealed that protein-protein interactions in the cytosol mediate the defined functions of TRIM proteins. For example, the ubiquitin E3 ligase enzymatic activity of several TRIM family members requires the B-box motif ...

Background: Nothing is known regarding the anti-apoptotic effect of HO-1 on hCSCs. Results: HO-1 expression induced by CoPP enhances hCSC survival through activation of the ERK/NRF2 signaling pathway and cytokine release. Conclusion: CoPP is a cytoprotective agent that could improve the efficacy of CSC-based therapies for heart disease. Significance: Strategies to enhance donor cell survival would have enormous therapeutic implications for patients with ischemic heart disease.

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3β (GSK-3β) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR−/−mice, IFNAR−/−-hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2–specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.