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Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues including wound healing, chronic ulcer and scaring. We previously identified a novel TRIM (tripartite-motif) family protein, named MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen over-production are observed in mg53-/- mice; and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. In vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation; the resulting down-regulation of alpha smooth muscle actin (α-SMA) and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a tri-functional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing.

Mitsugumin 29 (MG29), a member of the synaptophysin-like family proteins, is a transmembrane protein mainly expressed in the t-tubule membranes of skeletal muscle. Electron microscopy analysis of skeletal muscle derived from mg29-/- mice revealed morphological defects of t-tubules, indicating importance of MG29 in maintenance of muscle structure and function. To determine the function of MG29 in muscle physiology and regeneration, we performed co-immunoprecipitation and found that MG29 can bind to Bin1, another t-tubular protein.

Utilizing both overexpression and extracellular application, we aim to target MG53, an essential cell membrane repair protein, to valvular cells for treatment of tissue injuries associated with valvular heart disease (VHD). Membrane repair is important to maintain normal cellular physiology, and disruption of this process can result in disease in a number of different tissues. With the four heart valves experiencing a tremendous amount of mechanical stress, we have utilized microelectrode needle penetration and glass bead damage assays to assess the ability of MG53 to protect valvular interstitial cells (VICs) from physical injury. Furthermore, we have begun in vitro and in vivo studies to investigate the ability of MG53 to protect against the biological and physiological changes associated with VHD. To date, we have observed MG53 expression in valvular tissue and MG53-mediated protection against membrane damage through both live cell imaging and lactate dehydrogenase (LDH) release. Ongoing experiments are further characterizing mechanisms of protection against valvular fibrosis and calcification and assessing the effects of MG53 in in vivo models of VHD. Specifically, we have found that, in addition to protecting against cell membrane injury, recombinant human MG53 (rhMG53) can enter VICs and suppress TGF-beta-dependent activation of Smad and MAPK signaling associated with fibrocalcific valvular remodeling. We anticipate that, once all of our aims are completed, our experiments will greatly contribute to the study of cell biology, regenerative medicine, and cardiovascular disease, the number one cause of death in the United States and worldwide.

MG53 is a tripartite motif protein that is essential to plasma membrane regeneration. It binds to phosphatidylserine and facilitates the formation of repair patches at sites of plasma membrane disruption. Our previous studies have reported MG53's efficacy in ameliorating ischemia-reperfusion (I/R) injury to multiple organs including the heart, lungs, kidney, and brain. In I/R injury, mitochondria, however, are the primary victim of damage and the exposure of mitochondrial cardiolipin (CL) in response to I/R-induced oxidative stress procures cell death. Thus far, the role of MG53 in the preservation of mitochondrial function has not been studied. Here, we test the hypothesis that MG53 interacts with CL to protect mitochondria from oxidative stress. We find that mitochondria in the heart of MG53 knockout mouse are more susceptible to I/R injury than those of wild type mice. We also observe that MG53 targets mitochondria under chronic oxidative stress conditions, such as high-fat diet induced metabolic syndrome and amyotrophic lateral sclerosis. We demonstrate that MG53 binds to CL through a lipid dot-blot and a quantitative ELISA lipid-protein binding assay. Additionally, we show that exogenous recombinant human MG53 (rhMG53) protein can be up-taken by a variety of cells, including mesenchymal stem cells, human corneal epithelial cells, and valvular interstitial cells. Once internalized, rhMG53 translocates to mitochondria in response to oxidative stress induced by anoxia-reoxygenation treatment as revealed by live-cell imaging and 3D reconstruction of confocal microscopy. These data suggest that MG53 can target damaged mitochondria through a potential CL signaling mechanism and may implicate MG53 in mitochondrial membrane protection. To our knowledge, this study is the first to explore the interaction of MG53 with subcellular structures and provides a novel mechanism for MG53-mediated amelioration of I/R-induced tissue injury.

MG53 is a member of the tripartite motif family with E3 ubiquitin ligase activity, and plays important roles in cell membrane repair, tissue regeneration and wound healing. We previously showed that topical or systemic administration of recombinant human MG53 (rhMG53) protein protected against tissue injury and reduced the level of inflammatory cytokines (e.g. IL-1β) in circulation. NLRP3 is a key member of the inflammasome complex that mediates the cleavage and activation of caspase-1 for control of IL-1β release into circulation. As tissue injury is often associated with inflammation, we hypothesize that MG53 interacts with NLRP3 to control inflammasome signaling associated with tissue injury. With the mg53-/- mice, we found the spleen organ was significantly enlarged compared with the wild type littermates. The protein level of NLRP3 in the spleen was up-regulated in the mg53-/- mice. In cultured HEK293 cells, co-expression of MG53 with NLRP3 led to down-regulation of NLRP3 in a proteasome-dependent manner. Down-regulation of NLRP3 was not observed in cells co-transfected with a mutant MG53 lacking E3-ligase activity. Co-immunpprecipitation studies revealed a physical interaction between MG53 and NLRP3. With human monocytes (THP-1 cells) transfected with RFP-MG53 and GFP-NLRP3, we observed the trafficking of MG53 and NLRP3 associated with inflammasome formation. Upon treatment with LPS and ATP, THP-1 cells responded with formation of inflammasomes and co-segregation of NLRP3 and MG53. The level of NLRP3 in THP-1 cells co-expressing MG53 was significantly lower compared with cells do not express MG53. Together, these findings suggest that MG53 act as a negative regulator of NLRP3 to control inflammasome signaling. More in vivo studies are required to dissect the physiological role of MG53 in modulating the immune response associated with tissue injury.

MG53 is a member of tripartite motif (TRIM) family of proteins and an important component of the membrane repair machinery. In skeletal muscle, MG53 protects against eccentric contraction related damage, while in cardiac muscle MG53 protects against ischemia-reperfusion injury. While previous studies establish MG53 function in striated muscles, little is known about MG53 function in other tissues. Here we investigate the function of MG53 in the skin and its contribution to wound healing. We found that loss-of-function of MG53 caused delayed wound healing and defect in hair follicular structure with the mg53-/- mice. In vitro studies with expression of GFP-MG53 in cultured keratinocytes showed that the protein responded to membrane damage in the same fashion as in muscle fibers and other non-muscle cells. In vivo studies using rodent models of incisional and excisional wound demonstrated that both topical and subcutaneous application of recombinant human MG53 (rhMG53) protein could improve wound healing. For improvement of the efficacy of rhMG53 in wound healing, we used a formulation where rhMG53 is packaged into a hydrogel in liquid format at room temperature, which can polymerize upon exposure to the body temperature for controlled delivery of rhMG53 to the wound site. This hydrogel formulation significantly improved the efficacy for rhMG53 in healing of incisional wound. Our data suggest that MG53 has strong potential as a therapeutic agent for wound healing.

TRIC represents a novel class of trimeric intracellular cation channels located at the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) of multiple cell types. These channels regulate the permeability of K ions across the ER/SR and consequently the movement of Ca ions during excitation-contraction coupling. Previously we showed that genetic ablation of TRIC-A lead to compromised K-permeability and Ca release across the SR/ER membrane, supporting the hypothesis that TRIC could function as counter-ion channels that provide the flow of K ions into the SR during the acute phase of Ca release. In the absence of TRIC, overload of Ca inside the ER/SR causes instability of Ca storage and release, leading to stress-induced dysfunction of skeletal and cardiac muscles. Cardiomyocytes isolated from the wild type mice often show spontaneous Ca release during SR Ca overload, also called store-overload induced Ca release (SOICR) that is mediated by the type 2 ryanodine receptor (RyR2). The appearance of SOICR was significantly less frequent in the TRIC-A knockout cardiomyocytes. Biochemical studies revealed that the carboxyl-tail domain of TRIC-A could interact with the RyR channel, supporting the possibility that TRIC-A may directly regulate the Ca release activity. It has been shown that SOICR can be reconstituted in HEK293 cells expressing RyR2. We found that expression of TRIC-A in these RyR2-expressing HEK293 cells could prevent overload of Ca inside the ER and abolish SOICR generation. Together, our data suggest that functional interaction between TRIC-A and RyR can modulate the Ca release process from internal stores.

Calcium-induced-calcium-release (CICR) from the sarcoplasmic reticulum (SR) plays an integral role in excitation-contraction coupling - the driving mechanism behind synchronous cardiac muscle contractions. Alterations in CICR are commonly found in individuals suffering from cardiac arrhythmias, and closely associated with ventricular fibrillation, tachycardia, and sudden cardiac death. The ryanodine receptor 2 (RyR2) is a key mediator of CICR, functioning as a calcium channel along the SR. Elucidating the role of proteins that modulate RyR2-mediated CICR represents a major interest in cardiovascular research. We recently discovered a novel SR-resident membrane protein named mitsugumin 56 (MG56), which belongs to the membrane-bound o-acyltransferase (MBOAT) family of proteins. Knockout of MG56 produced a postnatal lethal phenotype. We have observed elevated Ca spark activity in MG56 null muscle fibers when compared with the wild type littermates. Using HEK293 cells with inducible RYR2 expression, we found that over-expression of MG56 led to suppression of RyR2-mediated store-overload induced calcium release (SOICR) from the endoplasmic reticulum (ER). The findings suggest the possibility that MG56 functions as a stabilizer of RyR channel function. Since MBOAT family proteins contain acyltransferase activity, we hypothesize that the putative acyltransferase function for MG56 may be involved in the Ca regulatory function. In preliminary studies, we found that pretreatment of muscle fibers with 2-bromohexanoic acid, an inhibitor of palmitoyl acyltransferases, led to suppression of Ca spark activity. The effect of 2- bromohexanoic acid was also observed in HEK293 cells expressing RyR2, where SOICR activity was reduced upon treatment. Future studies are required to dissect the functional interaction between MG56 and RyR2, and to delineate the potential role for MG56-mediated lipid modification of RyR2 in the overall Ca signaling in cardiac muscle.

Mistugumin 53 (MG53) is an essential molecule in facilitating cellular membrane repair. A member of the TRIM protein family, MG53 possesses a RING domain functioning as an E3 ligase to mediate the down-regulation of insulin receptor substrate-1 (IRS-1) in insulin signaling. Conflicting results have been presented that elevated MG53 expression may serve as a causative factor for the development of metabolic syndrome. This study was designed to test whether high-fat diet (HFD) treatment alters the expression and function of MG53 within mice models of metabolic syndrome. Western blotting showed that MG53 expression does not change within the skeletal and cardiac muscles of mice subjected to HFD treatment. This data contradicts earlier findings presented by Song et al. (Nature, 494: 375-379, 2013), who claimed that MG53 expression is markedly elevated in animal models of insulin resistance and metabolic syndrome. Rather, we found that stress provoked by metabolic syndrome in the mice models actually reduced MG53 levels in the serum. Immunohistochemical analyses revealed that skeletal muscle fibers of HFD-induced mice experience localization of intracellular MG53 around mitochondria. The reduced MG53 serum levels observed may explain the compromised tissue regenerative capacity of diabetic patients. Clustering of MG53 around mitochondria may represent an adaptive response to metabolic stress. Overall, our study supports the guardian function of MG53 in cell membrane repair and metabolic syndrome. Therapeutic approaches for an elevation of MG53 expression in tissues that bypasses its interaction with IRS-1 may be a novel approach to treating human diseases with degenerative tissue repair functions.

TRIC-A and TRIC-B are trimeric intracellular cation channels located at the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) of multiple cell types. These channels regulate the permeability of K ions across the SR/ER and consequently the movement of Ca ions during excitation-contraction coupling. Previously we showed that genetic ablation of TRIC led to compromised K-permeability and Ca release across the SR membrane, supporting the hypothesis that TRIC could function as counter-ion channels that allows the flow of K ions into the SR during the acute phase of Ca release. In the absence of TRIC, overload of Ca inside the ER/SR causes instability of Ca storage and release, leading to stress-induced dysfunction of multiple tissues. Spontaneous Ca waves, also called store overload-induced Ca release (SOICR) mediated by the type 2 ryanodine receptor (RyR2), evoke ventricular tachyarrhythmia in individuals with heart failure. Our biochemical studies revealed that the carboxyl-tail domain of TRIC-A could interact with the RyR channel, suggesting the possibility that TRIC-A may directly regulate the Ca release activity. We found that TRIC-A, but not TRIC-B, prevented the appearance of SOICR in HEK293 cells expressing RyR2. Cytosolic Ca measurement by Fura-2 and ER luminal Ca measurement by D1ER revealed that expression of TRIC-A in HEK293 cells could prevent overload of Ca inside the ER by targeting the RyR2 channel function. Such effect was translated into suppression of SOICR. These effects appeared to be specific for TRIC-A, as co-expression of RyR2 with TRIC-B did not affect SOICR. Together, our data suggest that functional interaction between TRIC-A and RyR can modulate the Ca release process from internal stores and regulate Ca homeostasis across the ER/SR.