Detection of Ras GTPase protein radicals through immuno-spin trapping

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward non-metalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of ·NO-mediated protein radical formation in the HRas GTPase. HRas is a non-metalloprotein that plays a critical role in regulating cell growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states where Ras GTPases play an important role

High-fat diet induces an initial adaptation of mitochondrial bioenergetics in the kidney despite evident oxidative stress and mitochondrial ROS production

Obesity and metabolic syndrome are associated with an increased risk for several diabetic complications, including diabetic nephropathy and chronic kidney diseases. Oxidative stress and mitochondrial dysfunction are often proposed mechanisms in various organs in obesity models, but limited data are available on the kidney. Here, we fed a lard-based high-fat diet to mice to investigate structural changes, cellular and subcellular oxidative stress and redox status, and mitochondrial biogenesis and function in the kidney. The diet induced characteristic changes, including glomerular hypertrophy, fibrosis, and interstitial scarring, which were accompanied by a proinflammatory transition. We demonstrate evidence for oxidative stress in the kidney through 3-nitrotyrosine and protein radical formation on high-fat diet with a contribution from iNOS and NOX-4 as well as increased generation of mitochondrial oxidants on carbohydrate- and lipid-based substrates. The increased H2O2 emission in the mitochondria suggests altered redox balance and mitochondrial ROS generation, contributing to the overall oxidative stress. No major derailments were observed in respiratory function or biogenesis, indicating preserved and initially improved bioenergetic parameters and energy production. We suggest that, regardless of the oxidative stress events, the kidney developed an adaptation to maintain normal respiratory function as a possible response to an increased lipid overload. These findings provide new insights into the complex role of oxidative stress and mitochondrial redox status in the pathogenesis of the kidney in obesity and indicate that early oxidative stress-related changes, but not mitochondrial bioenergetic dysfunction, may contribute to the pathogenesis and development of obesity-linked chronic kidney diseases

Myoglobin-H2O2 catalyzes the oxidation of beta-ketoacids to alpha-dicarbonyls: Mechanism and implications in ketosis

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H2O2. We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet alpha-dicarbonyl species (methylglyoxal and diacetyl). the observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 mu M) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an alpha-carbon-centered radical and acetyl radical. the latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA(center dot) adduct was observed and confirmed by isotope effects. Oxygen consumption and a-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H2O2 or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. the generation of radicals and triplet dicarbonyl products by Mb/H2O2/beta-ketoacids may contribute to the adverse health effects of ketogenic unbalance. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Ciencia e Tecnologia (INCT) RedoxomaNational Institute of Environmental Health Sciences (NIEHS)Universidade Federal de São Paulo, Dept Ciencias Exatas & Terra, Inst Ciencias Ambientais Quim & Farmaceut, BR-00972270 Diadema, SP, BrazilUniv São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, BrazilNatl Inst Environm Hlth Sci, Free Rad Metab Grp, Lab Toxicol & Pharmacol, NIH, Res Triangle Pk, NC 27709 USAUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, Inst Ciencias Ambientais Quim & Farmaceut, BR-00972270 Diadema, SP, BrazilWeb of Scienc