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.

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.

Functional mosaic organization of neuroligins in neuronal circuits.

Complex brain circuitry with feedforward and feedback systems regulates neuronal activity, enabling neural networks to process and drive the entire spectrum of cognitive, behavioral, sensory, and motor functions. Simultaneous orchestration of distinct cells and interconnected neural circuits is underpinned by hundreds of synaptic adhesion molecules that span synaptic junctions. Dysfunction of a single molecule or molecular interaction at synapses can lead to disrupted circuit activity and brain disorders. Neuroligins, a family of cell adhesion molecules, were first identified as postsynaptic-binding partners of presynaptic neurexins and are essential for synapse specification and maturation. Here, we review recent advances in our understanding of how this family of adhesion molecules controls neuronal circuit assembly by acting in a synapse-specific manner.

p55γ functional mimetic peptide N24 blocks vascular proliferative disorders.

UNLABELLED: Proliferation and migration disorders of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of proliferative cardiovascular diseases. Although, over the past two decades, a large panel of drugs has been developed for targeting VSMC proliferation, cardiovascular disease remains the leading cause of death worldwide. Thus, there is a compelling need to identify novel signaling pathways and molecules controlling VSMC proliferation and migration, to provide not only mechanistic insights but also safe and effective therapies for the treatment of cardiovascular diseases. Our recent studies have demonstrated that p55γ, a regulatory subunit of phosphoinositide 3-kinase, functions as an endogenous brake on VSMC proliferation. Here, we demonstrate that the small peptide N24, the first 24 amino acids of the NH2 terminus of p55γ, is a functional mimetic which negatively regulates VSMC proliferation and migration. Specifically, luminal delivery of adenovirus expressing N24 or local administration of Tat transactivator protein (TAT)-tagged N24 by pluronic gel alleviates neointimal formation following balloon injury in rat carotid arteries. Enforced expression of N24 suppresses the proliferation and migration of VSMCs induced by serum- or platelet-derived growth factor-BB. Mechanistically, N24 induces cell cycle arrest via activating the p53-p21 signal pathway, without triggering cell death. N24 interacts with and stabilizes p53 by blocking its ubiquitin-dependent degradation, subsequently promotes p21 transcription, and arrests cell cycle progression. Indeed, knockdown of p21 or p53 abrogates the N24-mediated cell growth arrest. Thus, N24 is a p55γ mimetic inhibiting VSMC proliferation as well as migration, thereby conferring important therapeutic implications for anti-proliferative treatment. KEY MESSAGE: • N24 attenuates balloon injury-induced neointimal formation. • Overexpression of N24 inhibits cultured VSMC proliferation and migration. • Overexpression of N24 arrests the cell cycle at S phase. • N24 interacts with and stabilizes p53 resulting in growth suppression.