S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death.
Oxidative stress and membrane damage following myocardial ischemia/reperfusion injury are important contributors to cardiomyocyte death and the loss of myocardial function. Our previous study identified cysteine 144 (C144) of tripartite motif-containing protein 72 (TRIM72) as a potential site for S-nitrosylation (SNO). TRIM72 is a cardioprotective membrane repair protein that can be both activated and targeted for degradation by different oxidative modifications. Consistent with the potential regulation of TRIM72 by various oxidative modifications, we found that SNO levels increased at C144 of TRIM72 with ischemic preconditioning. Therefore, to investigate the role of C144 in the regulation of TRIM72 function, we mutated C144 of TRIM72 to a serine residue (TRIM72(C144S)), and expressed either TRIM72(WT) or TRIM72(C144S) in HEK-293 cells, which lack endogenous TRIM72, in order to examine the effect of this mutation on the functional stability of TRIM72 and on cell survival. We hypothesized that SNO of TRIM72 stabilizes the protein, thus allowing for membrane repair and enhanced cell survival. Upon treatment with hydrogen peroxide (H2O2), we found that TRIM72(WT) levels were decreased, but not TRIM72(C144S) and this correlated with increased H2O2-induced cell death in TRIM72(WT) cells. Additionally, we found that treatment with the cardioprotective S-nitrosylating agent S-nitrosoglutathione (GSNO), was able to preserve TRIM72(WT) protein levels and enhance TRIM72(WT)-mediated cell survival, but had no effect on TRIM72(C144S) levels. Consistent with our hypothesis, GSNO was also found to increase SNO levels and inhibit H2O2-induced irreversible oxidation for TRIM72(WT) without affecting TRIM72(C144S). In further support of our hypothesis, GSNO blocked the ischemia/reperfusion-induced decrease in TRIM72 levels and reduced infarct size in a Langendorff-perfused heart model. The results of these studies have important implications for cardioprotection and suggest that SNO of TRIM72 at C144 prevents the oxidation-induced degradation of TRIM72 following oxidative insult, therefore enhancing cardiomyocyte survival.
Systems Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA; Division of Cardiovascular Pathology, Department of Pathology, 720 Rutland Avenue, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: mkohr1@jhmi.edu.
Mark J
MJ
Kohr
Systems Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA.
Alicia M
AM
Evangelista
Division of Cardiology, Department of Medicine, 720 Rutland Avenue, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Marcella
M
Ferlito
Division of Cardiovascular Pathology, Department of Pathology, 720 Rutland Avenue, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Charles
C
Steenbergen
Systems Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA.
Elizabeth
E
Murphy
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death; D002470:Cell Survival; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D019194:Ischemic Preconditioning; D008297:Male; D008565:Membrane Proteins; D008810:Mice, Inbred C57BL; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology* / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D059748:Proteolysis
D002352:Carrier Proteins; C538824:MG53 protein, mouse; D008565:Membrane Proteins; D009569:Nitric Oxide; D003545:Cysteine
Cardioprotection; Cell death; Mitsugumin 53; Oxidation; S-nitrosylation; Tripartite motif-containing protein 72