Negative regulation of AMPK signaling by high glucose via E3 ubiquitin ligase MG53.

As a master regulator of metabolism, AMP-activated protein kinase (AMPK) is activated upon energy and glucose shortage but suppressed upon overnutrition. Exaggerated negative regulation of AMPK signaling by nutrient overload plays a crucial role in metabolic diseases. However, the mechanism underlying the negative regulation is poorly understood. Here, we demonstrate that high glucose represses AMPK signaling via MG53 (also called TRIM72) E3-ubiquitin-ligase-mediated AMPKα degradation and deactivation. Specifically, high-glucose-stimulated reactive oxygen species (ROS) signals AKT to phosphorylate AMPKα at S485/491, which facilitates the recruitment of MG53 and the subsequent ubiquitination and degradation of AMPKα. In addition, high glucose deactivates AMPK by ROS-dependent suppression of phosphorylation of AMPKα at T172. These findings not only delineate the mechanism underlying the impairment of AMPK signaling in overnutrition-related diseases but also highlight the significance of keeping the yin-yang balance of AMPK signaling in the maintenance of metabolic homeostasis.

Targeting CaMKII-δ9 Ameliorates Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Inflammation.

BACKGROUND: CaMKII (Ca2+/calmodulin-dependent kinase II) plays a central role in cardiac ischemia/reperfusion (I/R) injury-an important therapeutic target for ischemic heart disease. In the heart, CaMKII-δ is the predominant isoform and further alternatively spliced into 11 variants. In humans, CaMKII-δ9 and CaMKII-δ3, the major cardiac splice variants, inversely regulate cardiomyocyte viability with the former pro-death and the latter pro-survival. However, it is unknown whether specific inhibition of the detrimental CaMKII-δ9 prevents cardiac I/R injury and, if so, what is the underlying mechanism. Here, we aim to investigate the cardioprotective effect of specific CaMKII-δ9 inhibition against myocardial I/R damage and determine the underlying mechanisms. METHODS: The role and mechanism of CaMKII-δ9 in cardiac I/R injury were investigated in mice in vivo, neonatal rat ventricular myocytes, and human embryonic stem cell-derived cardiomyocytes. RESULTS: We demonstrate that CaMKII-δ9 inhibition with knockdown or knockout of its feature exon, exon 16, protects the heart against I/R-elicited injury and subsequent heart failure. I/R-induced cardiac inflammation was also ameliorated by CaMKII-δ9 inhibition, and compared with the previously well-studied CaMKII-δ2, CaMKII-δ9 overexpression caused more profound cardiac inflammation. Mechanistically, in addition to IKKß (inhibitor of NF-κB [nuclear factor-κB] kinase subunit ß), CaMKII-δ9, but not δ2, directly interacted with IκBα (NF-κB inhibitor α) with its feature exon 13-16-17 combination and increased IκBα phosphorylation and consequently elicited more pronounced activation of NF-κB signaling and inflammatory response. Furthermore, the essential role of CaMKII-δ9 in myocardial inflammation and damage was confirmed in human cardiomyocytes. CONCLUSIONS: We not only identified CaMKII-δ9-IKK/IκB-NF-κB signaling as a new regulator of human cardiomyocyte inflammation but also demonstrated that specifically targeting CaMKII-δ9, the most abundant CaMKII-δ splice variant in human heart, markedly suppresses I/R-induced cardiac NF-κB activation, inflammation, and injury and subsequently ameliorates myocardial remodeling and heart failure, providing a novel therapeutic strategy for various ischemic heart diseases.

Blocking MG53S255 Phosphorylation Protects Diabetic Heart From Ischemic Injury.

BACKGROUND: As an integral component of cell membrane repair machinery, MG53 (mitsugumin 53) is important for cardioprotection induced by ischemia preconditioning and postconditioning. However, it also impairs insulin signaling via its E3 ligase activity-mediated ubiquitination-dependent degradation of IR (insulin receptor) and IRS1 (insulin receptor substrate 1) and its myokine function-induced allosteric blockage of IR. Here, we sought to develop MG53 into a cardioprotection therapy by separating its detrimental metabolic effects from beneficial actions. METHODS: Using immunoprecipitation-mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we investigated the role of MG53 phosphorylation at serine 255 (S255). In particular, utilizing recombinant proteins and gene knock-in approaches, we evaluated the potential therapeutic effect of MG53-S255A mutant in treating cardiac ischemia/reperfusion injury in diabetic mice. RESULTS: We identified S255 phosphorylation as a prerequisite for MG53 E3 ligase activity. Furthermore, MG53S255 phosphorylation was mediated by GSK3ß (glycogen synthase kinase 3 beta) and markedly elevated in the animal models with metabolic disorders. Thus, IR-IRS1-GSK3ß-MG53 formed a vicious cycle in the pathogenesis of metabolic disorders where aberrant insulin signaling led to hyper-activation of GSK3ß, which in turn, phosphorylated MG53 and enhanced its E3 ligase activity, and further impaired insulin sensitivity. Importantly, S255A mutant eliminated the E3 ligase activity while retained cell protective function of MG53. Consequently, the S255A mutant, but not the wild type MG53, protected the heart against ischemia/reperfusion injury in db/db mice with advanced diabetes, although both elicited cardioprotection in normal mice. Moreover, in S255A knock-in mice, S255A mutant also mitigated ischemia/reperfusion-induced myocardial damage in the diabetic setting. CONCLUSIONS: S255 phosphorylation is a biased regulation of MG53 E3 ligase activity. The MG53-S255A mutant provides a promising approach for the treatment of acute myocardial injury, especially in patients with metabolic disorders.

Novel CaMKII-δ Inhibitor Hesperadin Exerts Dual Functions to Ameliorate Cardiac Ischemia/Reperfusion Injury and Inhibit Tumor Growth.

BACKGROUND: Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart. METHODS: A small-molecule kinase inhibitor library and a high-throughput screening system for the kinase activity assay of CaMKII-δ9 (the most abundant CaMKII-δ splice variant in human heart) were used to screen for CaMKII-δ inhibitors. Using cultured neonatal rat ventricular myocytes, human embryonic stem cell-derived cardiomyocytes, and in vivo mouse models, in conjunction with myocardial injury induced by I/R (or hypoxia/reoxygenation) and CaMKII-δ9 overexpression, we sought to investigate the protection of hesperadin against cardiomyocyte death and cardiac diseases. BALB/c nude mice with xenografted tumors of human cancer cells were used to evaluate the in vivo antitumor effect of hesperadin. RESULTS: Based on the small-molecule kinase inhibitor library and screening system, we found that hesperadin, an Aurora B kinase inhibitor with antitumor activity in vitro, directly bound to CaMKII-δ and specifically blocked its activation in an ATP-competitive manner. Hesperadin functionally ameliorated both I/R- and overexpressed CaMKII-δ9-induced cardiomyocyte death, myocardial damage, and heart failure in both rodents and human embryonic stem cell-derived cardiomyocytes. In addition, in an in vivo BALB/c nude mouse model with xenografted tumors of human cancer cells, hesperadin delayed tumor growth without inducing cardiomyocyte death or cardiac injury. CONCLUSIONS: Here, we identified hesperadin as a specific small-molecule inhibitor of CaMKII-δ with dual functions of cardioprotective and antitumor effects. These findings not only suggest that hesperadin is a promising leading compound for clinical therapy of cardiac I/R injury and heart failure, but also provide a strategy for the joint therapy of cancer and cardiovascular disease caused by anticancer treatment.

Cryo-EM structure of human MG53 homodimer.

MG53 is a tripartite motif (TRIM) family E3 ligase and plays important biological functions. Here we present the cryo-EM structure of human MG53, showing that MG53 is a homodimer consisting of a 'body' and two 'wings'. Intermolecular interactions are mainly distributed in the 'body' which is relatively stable, while two 'wings' are more dynamic. The overall architecture of MG53 is distinct from those of TRIM20 and TRIM25, illustrating the broad structural diversity of this protein family.

Functional and Adaptive Significance of Promoter Mutations That Affect Divergent Myocardial Expressions of TRIM72 in Primates.

Cis-regulatory elements play important roles in tissue-specific gene expression and in the evolution of various phenotypes, and mutations in promoters and enhancers may be responsible for adaptations of species to environments. TRIM72 is a highly conserved protein that is involved in energy metabolism. Its expression in the heart varies considerably in primates, with high levels of expression in Old World monkeys and near absence in hominids. Here, we combine phylogenetic hypothesis testing and experimentation to demonstrate that mutations in promoter are responsible for the differences among primate species in the heart-specific expression of TRIM72. Maximum likelihood estimates of lineage-specific substitution rates under local-clock models show that relative to the evolutionary rate of introns, the rate of promoter was accelerated by 78% in the common ancestor of Old World monkeys, suggesting a role for positive selection in the evolution of the TRIM72 promoter, possibly driven by selective pressure due to changes in cardiac physiology after species divergence. We demonstrate that mutations in the TRIM72 promoter account for the differential myocardial TRIM72 expression of the human and the rhesus macaque. Furthermore, changes in TRIM72 expression alter the expression of genes involved in oxidative phosphorylation, which in turn affects mitochondrial respiration and cardiac energy capacity. On a broader timescale, phylogenetic regression analyses of data from 29 mammalian species show that mammals with high cardiac expression of TRIM72 have high heart rate, suggesting that the expression changes of TRIM72 may be related to differences in the heart physiology of those species.

Cardiac Ischemic Preconditioning Promotes MG53 Secretion Through H2O2-Activated Protein Kinase C-δ Signaling.

BACKGROUND: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (mitsugumin 53; also called TRIM72), not only plays an essential role in IPC-mediated cardioprotection against ischemia/reperfusion injury but also ameliorates mechanical damage. In addition to its intracellular actions, as a myokine/cardiokine, MG53 can be secreted from the heart and skeletal muscle in response to metabolic stress. However, it is unknown whether IPC-mediated cardioprotection is causally related to MG53 secretion and, if so, what the underlying mechanism is. METHODS: Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo, isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. Moreover, using recombinant MG53 proteins, we investigated the potential biological function of secreted MG53 in the context of IPC and ischemia/reperfusion injury. RESULTS: We found that IPC triggered robust MG53 secretion in rodents in vivo, perfused hearts, and cultured cardiac myocytes without causing cell membrane leakage. Mechanistically, IPC promoted MG53 secretion through H2O2-evoked activation of protein kinase-C-δ. Specifically, IPC-induced myocardial MG53 secretion was mediated by H2O2-triggered phosphorylation of protein kinase-C-δ at Y311, which is necessary and sufficient to facilitate MG53 secretion. Functionally, systemic delivery of recombinant MG53 proteins to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice but also protected rodent hearts from ischemia/reperfusion injury even in the absence of IPC. Moreover, oxidative stress by H2O2 augmented MG53 secretion, and MG53 knockdown exacerbated H2O2-induced cell injury in human embryonic stem cell-derived cardiomyocytes, despite relatively low basal expression of MG53 in human heart. CONCLUSIONS: We conclude that IPC and oxidative stress can trigger MG53 secretion from the heart via an H2O2-protein kinase-C-δ-dependent mechanism and that extracellular MG53 can participate in IPC protection against cardiac ischemia/reperfusion injury.

Glucose-Sensitive Myokine/Cardiokine MG53 Regulates Systemic Insulin Response and Metabolic Homeostasis.

BACKGROUND: Mitsugumin 53 (MG53 or TRIM72), a striated muscle-specific E3 ligase, promotes ubiquitin-dependent degradation of the insulin receptor and insulin receptor substrate-1 and subsequently induces insulin resistance, resulting in metabolic syndrome and type 2 diabetes mellitus (T2DM). However, it is unknown how MG53 from muscle regulates systemic insulin response and energy metabolism. Increasing evidence demonstrates that muscle secretes proteins as myokines or cardiokines that regulate systemic metabolic processes. We hypothesize that MG53 may act as a myokine/cardiokine, contributing to interorgan regulation of insulin sensitivity and metabolic homeostasis. METHODS: Using perfused rodent hearts or skeletal muscle, we investigated whether high glucose, high insulin, or their combination (conditions mimicking metabolic syndrome or T2DM) alters MG53 protein concentration in the perfusate. We also measured serum MG53 levels in rodents and humans in the presence or absence of metabolic diseases, particularly T2DM. The effects of circulating MG53 on multiorgan insulin response were evaluated by systemic delivery of recombinant MG53 protein to mice. Furthermore, the potential involvement of circulating MG53 in the pathogenesis of T2DM was assessed by neutralizing blood MG53 with monoclonal antibodies in diabetic db/db mice. Finally, to delineate the mechanism underlying the action of extracellular MG53 on insulin signaling, we analyzed the potential interaction of MG53 with extracellular domain of insulin receptor using coimmunoprecipitation and surface plasmon resonance assays. RESULTS: Here, we demonstrate that MG53 is a glucose-sensitive myokine/cardiokine that governs the interorgan regulation of insulin sensitivity. First, high glucose or high insulin induces MG53 secretion from isolated rodent hearts and skeletal muscle. Second, hyperglycemia is accompanied by increased circulating MG53 in humans and rodents with diabetes mellitus. Third, systemic delivery of recombinant MG53 or cardiac-specific overexpression of MG53 causes systemic insulin resistance and metabolic syndrome in mice, whereas neutralizing circulating MG53 with monoclonal antibodies has therapeutic effects in T2DM db/db mice. Mechanistically, MG53 binds to the extracellular domain of the insulin receptor and acts as an allosteric blocker. CONCLUSIONS: Thus, MG53 has dual actions as a myokine/cardiokine and an E3 ligase, synergistically inhibiting the insulin signaling pathway. Targeting circulating MG53 opens a new therapeutic avenue for T2DM and its complications.

Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders.

Insulin resistance is a fundamental pathogenic factor present in various metabolic disorders including obesity and type 2 diabetes. Although skeletal muscle accounts for 70-90% of insulin-stimulated glucose disposal, the mechanism underlying muscle insulin resistance is poorly understood. Here we show in mice that muscle-specific mitsugumin 53 (MG53; also called TRIM72) mediates the degradation of the insulin receptor and insulin receptor substrate 1 (IRS1), and when upregulated, causes metabolic syndrome featuring insulin resistance, obesity, hypertension and dyslipidaemia. MG53 expression is markedly elevated in models of insulin resistance, and MG53 overexpression suffices to trigger muscle insulin resistance and metabolic syndrome sequentially. Conversely, ablation of MG53 prevents diet-induced metabolic syndrome by preserving the insulin receptor, IRS1 and insulin signalling integrity. Mechanistically, MG53 acts as an E3 ligase targeting the insulin receptor and IRS1 for ubiquitin-dependent degradation, comprising a central mechanism controlling insulin signal strength in skeletal muscle. These findings define MG53 as a novel therapeutic target for treating metabolic disorders and associated cardiovascular complications.

MG53: Biological Function and Potential as a Therapeutic Target.

MG53 (also known as tripartite motif, TRIM72) is a cardiac and skeletal muscle-specific TRIM-family protein that exhibits multiple biologic functions. First, MG53 participates in plasma membrane repair of the heart, skeletal muscle, and, other tissues. Second, MG53 is essentially involved in the cardioprotection of cardiac ischemic, preconditioning, and postconditioning by activating the PI3K-Akt-GSK3ß and ERK1/2 survival signaling pathways. Moreover, systemic delivery of recombinant MG53 protein ameliorates the impact of a range of injury insults on the heart, skeletal muscle, lung, kidney, skin, and brain. It is noteworthy that chronic upregulation of MG53 induces insulin resistance and metabolic diseases, such as type 2 diabetes and its cardiovascular complications, by acting as an E3 ligase to mediate the degradation of insulin receptor and insulin receptor substrate-1. In addition, MG53 negatively regulates myogenesis. In summary, MG53 is a multifunctional protein involved in the vital physiologic and pathologic processes of multiple organs and is a promising therapeutic target for various human diseases. In this review, we comprehensively summarize current research progress on the biologic functions and therapeutic potential of MG53.

Low-cost dynamic real-time foveated imager.

Foveated imaging systems have the ability to capture local high-resolution or high-magnification images with wide field of view (FOV); thus, they have great potential for applications in the field of monitoring and remote sensing of unmanned aerial vehicles. Hence, foveated optical systems are in strong demand. However, the existing foveated imaging systems either are equipped with expensive modulators or require fixing the local high resolution imaging field, which is not suitable for mass production or object tracking in industrial applications. We propose a low-cost dynamic real-time foveated imaging system for extensive use in the listed applications. Specifically, we place a microlens behind the first intermediary image plane to modulate the local focal length, constructing a local high magnification imaging channel. One two-axis translation stage drives the microlens to scan in the plane perpendicular to the optical axis, resulting in dynamic local high magnifying imaging. Furthermore, the peripheral imaging channel and the foveated imaging channel focus on the same detector, and the post image fusion is unnecessary; the system consists of only a common aspherical lens and thus is very inexpensive. The experimental system has a focal length of 25 mm, a full FOV of 30°, and an entrance pupil diameter of 5 mm, while the local high magnifying imaging channel has a focal length of 35 mm and FOV of 15°. Experiment results show that the low-cost dynamic real-time foveated imaging system performs very well.

MG53 is a double-edged sword for human diseases.

Mitsugumin 53 (MG53), also named Trim72, is a multi-functional TRIM-family protein, which is abundantly expressed in cardiac and skeletal muscle. It has been shown that MG53 not only plays important physiological roles but also acts as a crucial pathogenic factor of various diseases. First, MG53 preserves cardiac and skeletal muscle integrity via facilitating plasma membrane repair. Second, MG53 is essentially involved in cardiac ischemic preconditioning and postconditioning by activating PI3K-Akt-GSK3ß and ERK1/2 cell survival signaling pathways. Moreover, systemic delivery of recombinant MG53 is able to abolish mechanic or ischemia-reperfusion (I/R)-induced injury of multiple organs, including heart, skeletal muscle, lung, kidney and skin. Importantly, MG53 acts as an E3 ligase to mediate the degradation of insulin receptor and insulin receptor substrate-1, and subsequently induces insulin resistance and metabolic diseases such as type-2 diabetes and its cardiovascular complications. In addition, MG53 negatively regulates myogenesis. As a potential therapeutic target of human diseases, multiple facets of MG53 biological function and mechanisms of action should be taken into the consideration to maximize its beneficial effects and minimize potential side-effects. Here in this review, we intend to comprehensively summarize the current progresses on the biological functions of MG53, focusing on its clinical value as a therapeutic target.

Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation.

The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3' end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5' end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5' and 3' mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs.

Activation of GPR81 by lactate drives tumour-induced cachexia.

Cachexia affects 50-80% of patients with cancer and accounts for 20% of cancer-related death, but the underlying mechanism driving cachexia remains elusive. Here we show that circulating lactate levels positively correlate with the degree of body weight loss in male and female patients suffering from cancer cachexia, as well as in clinically relevant mouse models. Lactate infusion per se is sufficient to trigger a cachectic phenotype in tumour-free mice in a dose-dependent manner. Furthermore, we demonstrate that adipose-specific G-protein-coupled receptor (GPR)81 ablation, similarly to global GPR81 deficiency, ameliorates lactate-induced or tumour-induced adipose and muscle wasting in male mice, revealing adipose GPR81 as the major mediator of the catabolic effects of lactate. Mechanistically, lactate/GPR81-induced cachexia occurs independently of the well-established protein kinase A catabolic pathway, but it is mediated by a signalling cascade sequentially activating Gi-Gßγ-RhoA/ROCK1-p38. These findings highlight the therapeutic potential of targeting GPR81 for the treatment of this life-threatening complication of cancer.

Glycogen synthase kinase 3ß (GSK3ß) modulates antiviral activity of zinc-finger antiviral protein (ZAP).

Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including HIV-1, Ebola virus, and Sindbis virus. ZAP binds directly to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. ZAP has also been suggested to repress translation of the target mRNA. In this study, we report that ZAP is phosphorylated by glycogen synthase kinase 3ß (GSK3ß). GSK3ß sequentially phosphorylated Ser-270, Ser-266, Ser-262, and Ser-257 of rat ZAP. Inhibition of GSK3ß by inhibitor SB216763 or down-regulation of GSK3ß by RNAi reduced the antiviral activity of ZAP. These results indicate that phosphorylation of ZAP by GSK3ß modulates ZAP activity.

E3 ligase MG53 suppresses tumor growth by degrading cyclin D1.

Due to the essential role of cyclin D1 in regulating transition from G1 to S phase in cell cycle, aberrant cyclin D1 expression is a major oncogenic event in many types of cancers. In particular, the dysregulation of ubiquitination-dependent degradation of cyclin D1 contributes to not only the pathogenesis of malignancies but also the refractory to cancer treatment regiments with CDK4/6 inhibitors. Here we show that in colorectal and gastric cancer patients, MG53 is downregulated in more than 80% of tumors compared to the normal gastrointestinal tissues from the same patient, and the reduced MG53 expression is correlated with increased cyclin D1 abundance and inferior survival. Mechanistically, MG53 catalyzes the K48-linked ubiquitination and subsequent degradation of cyclin D1. Thus, increased expression of MG53 leads to cell cycle arrest at G1, and thereby markedly suppresses cancer cell proliferation in vitro as well as tumor growth in mice with xenograft tumors or AOM/DSS induced-colorectal cancer. Consistently, MG53 deficiency results in accumulation of cyclin D1 protein and accelerates cancer cell growth both in culture and in animal models. These findings define MG53 as a tumor suppressor via facilitating cyclin D1 degradation, highlighting the therapeutic potential of targeting MG53 in treating cancers with dysregulated cyclin D1 turnover.

MG53 E3 Ligase-Dead Mutant Protects Diabetic Hearts From Acute Ischemic/Reperfusion Injury and Ameliorates Diet-Induced Cardiometabolic Damage.

Cardiometabolic diseases, including diabetes and its cardiovascular complications, are the global leading causes of death, highlighting a major unmet medical need. Over the past decade, mitsugumin 53 (MG53), also called TRIM72, has emerged as a powerful agent for myocardial membrane repair and cardioprotection, but its therapeutic value is complicated by its E3 ligase activity, which mediates metabolic disorders. Here, we show that an E3 ligase-dead mutant, MG53-C14A, retains its cardioprotective function without causing metabolic adverse effects. When administered in normal animals, both the recombinant human wild-type MG53 protein (rhMG53-WT) and its E3 ligase-dead mutant (rhMG53-C14A) protected the heart equally from myocardial infarction and ischemia/reperfusion (I/R) injury. However, in diabetic db/db mice, rhMG53-WT treatment markedly aggravated hyperglycemia, cardiac I/R injury, and mortality, whereas acute and chronic treatment with rhMG53-C14A still effectively ameliorated I/R-induced myocardial injury and mortality or diabetic cardiomyopathy, respectively, without metabolic adverse effects. Furthermore, knock-in of MG53-C14A protected the mice from high-fat diet-induced metabolic disorders and cardiac damage. Thus, the E3 ligase-dead mutant MG53-C14A not only protects the heart from acute myocardial injury but also counteracts metabolic stress, providing a potentially important therapy for the treatment of acute myocardial injury in metabolic disorders, including diabetes and obesity.

MG53 constitutes a primary determinant of cardiac ischemic preconditioning.

BACKGROUND: Ischemic heart disease is the greatest cause of death in Western countries. The deleterious effects of cardiac ischemia are ameliorated by ischemic preconditioning (IPC), in which transient ischemia protects against subsequent severe ischemia/reperfusion injury. IPC activates multiple signaling pathways, including the reperfusion injury salvage kinase pathway (mainly PI3K-Akt-glycogen synthase kinase-3beta [GSK3beta] and ERK1/2) and the survivor activating factor enhancement pathway involving activation of the JAK-STAT3 axis. Nevertheless, the fundamental mechanism underlying IPC is poorly understood. METHODS AND RESULTS: In the present study, we define MG53, a muscle-specific TRIM-family protein, as a crucial component of cardiac IPC machinery. Ischemia/reperfusion or hypoxia/oxidative stress applied to perfused mouse hearts or neonatal rat cardiomyocytes, respectively, causes downregulation of MG53, and IPC can prevent ischemia/reperfusion-induced decrease in MG53 expression. MG53 deficiency increases myocardial vulnerability to ischemia/reperfusion injury and abolishes IPC protection. Overexpression of MG53 attenuates whereas knockdown of MG53 enhances hypoxia- and H(2)O(2)-induced cardiomyocyte death. The cardiac protective effects of MG53 are attributable to MG53-dependent interaction of caveolin-3 with phosphatidylinositol 3 kinase and subsequent activation of the reperfusion injury salvage kinase pathway without altering the survivor activating factor enhancement pathway. CONCLUSIONS: These results establish MG53 as a primary component of the cardiac IPC response, thus identifying a potentially important novel therapeutic target for the treatment of ischemic heart disease.

p72 DEAD box RNA helicase is required for optimal function of the zinc-finger antiviral protein.

The zinc-finger antiviral protein (ZAP) specifically inhibits the replication of many viruses by preventing the accumulation of viral mRNAs in the cytoplasm. ZAP directly binds to the viral mRNAs and recruits the RNA exosome to degrade the target RNA. In the present study, we identified the p72 DEAD box RNA helicase, but not the highly similar RNA helicase p68, as a ZAP-interacting protein. The binding domain of ZAP was mapped to its N-terminal portion, whereas both the N- and C-terminal domains of p72 bound to ZAP. Overexpression of the C-terminal domain of p72 reduced ZAP's activity, whereas overexpression of the full-length p72 enhanced ZAP's activity. The RNA helicase activity was required for p72 to promote ZAP-mediated RNA degradation. Depletion of p72 by RNAi also reduced ZAP's activity but did not affect tristetraprolin-mediated RNA degradation. We conclude that p72 is required for the optimal activity of ZAP, and we propose that p72 helps to restructure the ZAP-bound target mRNA for efficient degradation.