Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility.

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

Supplementation with omega-3 fatty acids potentiates oxidative stress in human airway epithelial cells exposed to ozone.

BACKGROUND: Dietary intake of the omega-3 family of polyunsaturated fatty acids (ω-3 FA) is associated with anti-inflammatory effects. However, unsaturated fatty acids are susceptible to oxidation, which produces pro-inflammatory mediators. Ozone (O3) is a tropospheric pollutant that reacts rapidly with unsaturated fatty acids to produce electrophilic and oxidative mediators of inflammation. OBJECTIVE: Determine whether supplementation with ω-3 FA alters O3-induced oxidative stress in human airway epithelial cells (HAEC). METHODS: 16-HBE cells expressing a genetically encoded sensor of the reduced to oxidized glutathione ratio (GSH/GSSG, EGSH) were supplemented with saturated, monounsaturated, or ω-3 FA prior to exposure to 0, 0.08, 0.1, or 0.3 ppm O3. Lipid peroxidation was measured in cellular lipid extracts and intact cells following O3 exposure. RESULTS: Relative to cells incubated with the saturated or monounsaturated fatty acids, cells supplemented with ω-3 FA containing 5 or 6 double bonds showed a marked increase in EGSH during exposure to O3 concentrations as low as 0.08 ppm. Consistent with this finding, the concentration of lipid hydroperoxides produced following O3 exposure was significantly elevated in ω-3 FA supplemented cells. DISCUSSION: Supplementation with polyunsaturated ω-3 FA potentiates oxidative responses, as indicated by EGSH, in HAEC exposed to environmentally relevant concentrations of O3. This effect is mediated by the increased formation of lipid hydroperoxides produced by the reaction of O3 with polyunsaturated fatty acids. Given the inflammatory activity of lipid hydroperoxides, these findings have implications for the potential role of ω-3 FA in increasing human susceptibility to the adverse health effects of O3 exposure.

Long chain lipid hydroperoxides increase the glutathione redox potential through glutathione peroxidase 4.

BACKGROUND: Peroxidation of PUFAs by a variety of endogenous and xenobiotic electrophiles is a recognized pathophysiological process that can lead to adverse health effects. Although secondary products generated from peroxidized PUFAs have been relatively well studied, the role of primary lipid hydroperoxides in mediating early intracellular oxidative events is not well understood. METHODS: Live cell imaging was used to monitor changes in glutathione (GSH) oxidation in HAEC expressing the fluorogenic sensor roGFP during exposure to 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HpODE), a biologically important long chain lipid hydroperoxide, and its secondary product 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). The role of hydrogen peroxide (H2O2) was examined by direct measurement and through catalase interventions. shRNA-mediated knockdown of glutathione peroxidase 4 (GPx4) was utilized to determine its involvement in the relay through which 9-HpODE initiates the oxidation of GSH. RESULTS: Exposure to 9-HpODE caused a dose-dependent increase in GSH oxidation in HAEC that was independent of intracellular or extracellular H2O2 production and was exacerbated by NADPH depletion. GPx4 was involved in the initiation of GSH oxidation in HAEC by 9-HpODE, but not that induced by exposure to H2O2 or the low molecular weight alkyl tert-butyl hydroperoxide (TBH). CONCLUSIONS: Long chain lipid hydroperoxides can directly alter cytosolic EGSH independent of secondary lipid oxidation products or H2O2 production. NADPH has a protective role against 9-HpODE induced EGSH changes. GPx4 is involved specifically in the reduction of long-chain lipid hydroperoxides, leading to GSH oxidation. SIGNIFICANCE: These results reveal a previously unrecognized consequence of lipid peroxidation, which may provide insight into disease states involving lipid peroxidation in their pathogenesis.

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components.

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H2O2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H2O2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.

Description of Immundisolibacter cernigliae gen. nov., sp. nov., a high-molecular-weight polycyclic aromatic hydrocarbon-degrading bacterium within the class Gammaproteobacteria, and proposal of Immundisolibacterales ord. nov. and Immundisolibacteraceae fam. nov.

The bacterial strain TR3.2T was isolated from aerobic bioreactor-treated soil from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Salisbury, NC, USA. Strain TR3.2T was identified as a member of 'Pyrene Group 2' or 'PG2', a previously uncultivated cluster of organisms associated with the degradation of high-molecular-weight PAHs by stable-isotope probing. Based on its 16S rRNA gene sequence, the strain was classified as a member of the class Gammaproteobacteria but possessed only 90.5 % gene identity to its closest described relative, Methylococcus capsulatus strain Bath. Strain TR3.2T grew on the PAHs pyrene, phenanthrene, anthracene, benz[a]anthracene and fluorene, as well as the azaarene carbazole, and could additionally metabolize a limited number of organic acids. Optimal growth occurred aerobically under mesophilic temperature, neutral pH and low salinity conditions. Strain TR3.2T was catalase and oxidase positive. Predominant fatty acids were C17 : 0 cyclo and C16 : 0. Genomic G+C content of the single chromosome was 67.79 mol% as determined by complete genome sequencing. Due to the high sequence divergence from any cultivated species and its unique physiological properties compared to its closest relatives, strain TR3.2T is proposed as a representative of a novel order, family, genus and species within the class Gammaproteobacteria, for which the name Immundisolibacter cernigliae gen. nov., sp. nov. is proposed. The associated order and family are therefore proposed as Immundisolibacteralesord. nov. and Immundisolibacteraceaefam. nov. The type strain of the species is TR3.2T (=ATCC TSD-58T=DSM 103040T).

Rugosibacter aromaticivorans gen. nov., sp. nov., a bacterium within the family Rhodocyclaceae, isolated from contaminated soil, capable of degrading aromatic compounds.

A bacterial strain designated Ca6T was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil from the site of a former manufactured gas plant in Charlotte, NC, USA, and linked phylogenetically to the family Rhodocyclaceae of the class Betaproteobacteria. Its 16S rRNA gene sequence was highly similar to globally distributed environmental sequences, including those previously designated 'Pyrene Group 1' demonstrated to grow on the PAHs phenanthrene and pyrene by stable-isotope probing. The most closely related described relative was Sulfuritalea hydrogenivorans strain sk43HT (93.6 % 16S rRNA gene sequence identity). In addition to a limited number of organic acids, Ca6T was capable of growth on the monoaromatic compounds benzene and toluene, and the azaarene carbazole, as sole sources of carbon and energy. Growth on the PAHs phenanthrene and pyrene was also confirmed. Optimal growth was observed aerobically under mesophilic temperature, neutral pH and low salinity conditions. Major fatty acids present included summed feature 3 (C16 : 1ω7c or C16 : 1ω6c) and C16 : 0. The DNA G+C content of the single chromosome was 55.14  mol% as determined by complete genome sequencing. Due to its distinct genetic and physiological properties, strain Ca6T is proposed as a member of a novel genus and species within the family Rhodocyclaceae, for which the name Rugosibacter aromaticivorans gen. nov., sp. nov. is proposed. The type strain of the species is Ca6T (=ATCC TSD-59T=DSM 103039T).