Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Time-course analysis of Streptococcus sanguinis after manganese depletion reveals changes in glycolytic, nucleotide, and redox metabolites

IntroductionManganese is important for the endocarditis pathogen, Streptococcus sanguinis. Little is known about why manganese is required for virulence or how it impacts the metabolome of streptococci.ObjectivesWe applied untargeted metabolomics to cells and media to understand temporal changes resulting from manganese depletion.MethodsEDTA was added to a S. sanguinis manganese-transporter mutant in aerobic fermentor conditions. Cell and media samples were collected pre- and post-EDTA treatment. Metabolomics data were generated using positive and negative modes of data acquisition on an LC-MS/MS system. Data were subjected to statistical processing using MetaboAnalyst and time-course analysis using Short Time series Expression Miner (STEM).ResultsWe observed quantitative changes in 534 and 422 metabolites in cells and media, respectively, after EDTA addition. The 173 cellular metabolites identified as significantly different indicated enrichment of purine and pyrimidine metabolism. Further multivariate analysis revealed that the top 15 cellular metabolites belonged primarily to lipids and redox metabolites. The STEM analysis revealed global changes in cells and media in comparable metabolic pathways. Products of glycolysis such as pyruvate and fructose-1,6-bisphosphate increased, suggesting that enzymes that act on them may require manganese for activity or expression. Nucleosides accumulated, possibly due to a blockage in conversion to nucleobases. Simultaneous accumulation of ortho-tyrosine and reduced glutathione suggests that cells were unable to utilize glutathione as a reductant.ConclusionDifferential analysis of metabolites revealed the activation of a number of metabolic pathways in response to manganese depletion, many of which may be connected to carbon catabolite repression.

Nicotinamide adenine dinucleotide (NAD+): essential redox metabolite, co-substrate and an anti-cancer and anti-ageing therapeutic target

Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH are essential coupled redox metabolites that primarily promote cellular oxidative (catabolic) metabolic reactions. This enables energy generation through glycolysis and mitochondrial respiration to support cell growth and survival. In addition, many key enzymes that regulate diverse cell functions ranging from gene expression to proteostasis require NAD+ as a co-substrate for their catalytic activity. This includes the NAD+-dependent sirtuin family of protein deacetylases and the PARP family of DNA repair enzymes. Whilst their vital activity consumes NAD+ which is cleaved to nicotinamide, several pathways exist for re-generating NAD+ and sustaining NAD+ homeostasis. However, there is growing evidence of perturbed NAD+ homeostasis and NAD+-regulated processes contributing to multiple disease states. NAD+ levels decline in the human brain and other organs with age and this is associated with neurodegeneration and other age-related diseases. Dietary supplementation with NAD+ precursors is being investigated to counteract this. Paradoxically, many cancers have increased dependency on NAD+. Clinical efforts to exploit this have so far shown limited success. Emerging new opportunities to exploit dysregulation of NAD+ metabolism in cancers are critically discussed. An update is also provided on other key NAD+ research including perturbation of the NAD+ salvage enzyme NAMPT in the context of the tumour microenvironment (TME), methodology to study subcellular NAD+ dynamics in real-time and the regulation of differentiation by competing NAD+ pools.

A novel redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

Aberrant regulation of metabolic kinases by altered redox homeostasis is a major contributing factor in aging and disease such as diabetes. However, the biochemical mechanisms by which metabolic kinases are regulated under oxidative stress is poorly understood. In this study, we demonstrate that the catalytic activity of a conserved family of Fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases (ePKs), are regulated by redox-active cysteines in the kinase domain. By solving the crystal structure of FN3K homolog from Arabidopsis thaliana (AtFN3K), we demonstrate that it forms an unexpected strand-exchange dimer in which the ATP binding P-loop and adjoining beta strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained inter-chain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirm that the strained disulfides function as redox “switches” to reversibly regulate FN3K activity and dimerization. Consistently, we find that human FN3K (HsFN3K), which contains an equivalent P-loop Cys, is also redox-sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, are not. Furthermore, CRISPR knockout of FN3K in human HepG2 cells results in significant upregulation of redox metabolites including glutathione. We propose that redox regulation evolved progressively in FN3Ks in response to changing cellular redox conditions. Our studies provide important new insights into the origin and evolution of redox regulation in the protein kinase superfamily and open new avenues for targeting HsFN3K in diabetic complications.

NADPH and glutathione redox link TCA cycle activity to endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is associated with dysregulated metabolism, but little is known about how the ER responds to metabolic activity. Here, working primarily in mouse hepatocytes, we show that decreasing the availability of substrate for the TCA cycle diminished NADPH production and attenuated ER stress in a manner that depended on glutathione oxidation. ER stress was also alleviated by impairing either TCA-dependent NADPH production or Glutathione Reductase. Conversely, stimulating TCA activity favored NADPH production, glutathione reduction, and ER stress. Validating these findings, we show that deletion of the mitochondrial pyruvate carrier, which is known to decrease TCA cycle activity and protect the liver from diet-induced injury, also diminished NADPH, elevated glutathione oxidation, and alleviated ER stress. These results provide independent genetic evidence that mitochondrial oxidative metabolism is linked to ER homeostasis. Our results demonstrate a novel pathway of communication between mitochondria and the ER, through relay of redox metabolites.

Redox homeostasis in the growth zone of the rice leaf plays a key role in cold tolerance

We analysed the cellular and molecular changes in the leaf growth zone of tolerant and sensitive rice varieties in response to suboptimal temperatures. Cold reduced the final leaf length by 35% and 51% in tolerant and sensitive varieties, respectively. Tolerant lines exhibited a smaller reduction of the leaf elongation rate and greater compensation by an increased duration of leaf growth. Kinematic analysis showed that cold reduced cell production in the meristem and the expansion rate in the elongation zone, but the latter was compensated for by a doubling of the duration of cell expansion. We performed iTRAQ proteome analysis on proliferating and expanding parts of the leaf growth zone. We identified 559 and 542 proteins, of which 163 and 210 were differentially expressed between zones, and 96 and 68 between treatments, in the tolerant and sensitive lines, respectively. The categories protein biosynthesis and redox homeostasis were significantly overrepresented in the up-regulated proteins. We therefore measured redox metabolites and enzyme activities in the leaf growth zone, demonstrating that tolerance of rice lines to suboptimal temperatures correlates with the ability to up-regulate enzymatic antioxidants in the meristem and non-enzymatic antioxidants in the elongation zone.