Regulation by mitochondrial superoxide and NADPH oxidase of cellular formation of nitrated cyclic GMP: potential implications for ROS signaling

8-Nitro-cGMP (8-nitroguanosine 3′,5′-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H 2O 2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H 2O 2. Using the mitochondria-targeted chemical probe MitoSOX™ Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H 2O 2regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H 2O 2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger thatmay be implicated in regulating ROS signalling in the presence of NO

Association of Uncoupling Protein 1 (UCP1) gene polymorphism with obesity: a case-control study

Abstract Background Obesity is one of the main causes of morbidity and mortality worldwide. More than 120 genes have been shown to be associated with obesity related phenotypes. The aim of this study was to determine the effect of selected genetic polymorphisms in Uncoupling protein 1 (UCP1) and Niemann-Pick C1 (NPC1) genes in an obese population in Saudi Arabia. Methods The genotypes of rs1800592, rs10011540 and rs3811791 (UCP1 gene) and rs1805081 and rs1805082 (NPC1 gene) were determined in a total of 492 subjects using TaqMan chemistry by Real-time PCR. In addition, capillary sequencing assay was performed to identify two specific polymorphisms viz., rs45539933 (exon 2) and rs2270565 (exon 5) of UCP1 gene. Results A significant association of UCP1 polymorphisms rs1800592 [OR, 1.52 (1.10–2.08); p = 0.009] was observed in the obese cohort after adjusting with age, sex and type 2 diabetes. Further BMI based stratification revealed that this association was inconsistent with both moderate and extreme obese cohort. A significant association of UCP1 polymorphisms rs3811791 was observed only in the moderate-obese cohort [OR = 2.89 (1.33–6.25); p = 0.007] but not in the extreme-obese cohort indicating an overlying genetic complexity between moderate-obesity and extreme-obesity. The risk allele frequencies, which were higher in moderate-obese cohort, had abnormal HDL, LDL and triglyceride levels. Conclusion The rs1800592 and rs3811791 of UCP1 gene are associated with obesity in general and in the moderate-obese group in particular. The associated UCP1 polymorphisms in the moderate-obese group may regulate the impaired energy metabolism which plays a significant role in the initial stages of obesity

Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein <i>S</i>‑Guanylation

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of guanosine 3′,5′-cyclic monophosphate (cGMP) formed endogenously under conditions associated with production of both reactive oxygen species and nitric oxide. It acts as an electrophilic second messenger in the regulation of cellular signaling by inducing a post-translational modification of redox-sensitive protein thiols via covalent adduction of cGMP moieties to protein thiols (protein <i>S</i>-guanylation). Here, we demonstrate that 8-nitro-cGMP potentially <i>S</i>-guanylates thiol groups of cGMP-dependent protein kinase (PKG), the enzyme that serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses. <i>S</i>-Guanylation of PKG was found to occur in a site specific manner; Cys42 and Cys195 were the susceptible residues among 11 Cys residues. Importantly, <i>S</i>-guanylation at Cys195, which is located in the high-affinity cGMP binding domain of PKG, causes persistent enzyme activation as determined by <i>in vitro</i> kinase assay as well as by an organ bath assay. <i>In vivo</i>, <i>S</i>-guanylation of PKG was demonstrated to occur in mice without any specific treatment and was significantly enhanced by lipopolysaccharide administration. These findings warrant further investigation in terms of the physiological and pathophysiological roles of <i>S</i>-guanylation-dependent persistent PKG activation