Blocking MG53 ^S255 Phosphorylation Protects Diabetic Heart From Ischemic Injury

Abstract

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, MG53 S255 phosphorylation was mediated by GSK3β (glycogen synthase kinase 3 beta) and markedly elevated in the animal models with metabolic disorders. Thus, IR-IRS1-GSK3β-MG53 formed a vicious cycle in the pathogenesis of metabolic disorders where aberrant insulin signaling led to hyper-activation of GSK3β, 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.

Prohibiting MG53 Phosphorylation Optimizes its Therapeutic Potential in Diabetes

“…In summary, the study by Lv et al 14 provides new insights into how MG53 targets protein substrates for ubiquitination. In diabetic heart, GSK3-dependent phosphorylation of MG53 at Serine 255 increases MG53 recognition, ubiquitination, and degradation of critical insulin signaling pathway proteins, contributing to insulin resistance and increased susceptibility to ischemic injury.…”

“…In this issue, Lv et al 14 extended their findings into MG53 ligase function by exploring whether endogenous kinases act to shift the balance between MG53 substrate ubiquitination and membrane healing activities (Figure). By performing mass spectrometry analysis on MG53 immunoprecipitated from skeletal myocytes and heterologously expressing HEK cells, the residue Serine 255 was found to be phosphorylated in both groups and among 5 phosphosites in the muscle expressed protein.…”

GSK3β: A ray of hope for the treatment of diabetic kidney disease

“…In particular, GSK3β is a kinase required for high glucose-induced serine332 phosphorylation, ubiquitination, and degradation of IRS1. 29 Likewise, GSK3βenhanced phosphorylation of MG53 enables its E3 ligase activity to mediate the degradation of both IR and IRS1, 30 which may partly account for the involvement of GSK3β in insulin resistance. Moreover, Chunyi et al revealed that augmented GSK3β phosphorylation elevates AKT signaling in breast cancer.…”

MG53/TRIM72: multi-organ repair protein and beyond

“…For instance, investigations have successfully attenuated insulin resistance induced by MG53 through targeted disruption of the interaction between MG53 and IRS-1 (Lee et al, 2016). The MG53 mutant currently under construction exhibits the ability to circumvent its E3 ubiquitin ligase activity (Lv et al, 2022), thereby offering a novel therapeutic approach for diabetes, muscle injury diseases, and cardiovascular disorders.…”

“…On this basis, efforts should be made to preserve the anti-tumor therapeutic ability of MG53 while mitigating its side effects. Currently, MG53 mutants, which retain membrane repair function without compromising glucose metabolism, have been developed by eliminating the E3 ubiquitin ligase properties of MG53 (Lv et al, 2022). However, in the context of cancer treatment, the E3 ubiquitin ligase of MG53 appears to play a pivotal role.…”