title: Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction.
abstract: Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.
authors: From the Department of Anesthesiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Jaganathan
authors: J
authors: Subramani
authors: From the Department of Anesthesiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Venkatesh
authors: V
authors: Kundumani-Sridharan
authors: From the Department of Anesthesiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Rob H P
authors: RH
authors: Hilgers
authors: From the Department of Anesthesiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.
authors: Cade
authors: C
authors: Owens
authors: From the Department of Anesthesiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 kumuda.das@ttuhsc.edu.
authors: Kumuda C
authors: KC
authors: Das
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology; D005978:Glutathione / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D009203:Myocardial Infarction / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D034741:RNA, Small Interfering; D013879:Thioredoxins; D052250:Nitric Oxide Synthase Type III; D005978:Glutathione
keywords: endothelial dysfunction; glutathionylation; heart failure; ischemia; myocardial infarction; nitric-oxide synthase; reperfusion; thioredoxin; thioredoxin reductase