Oxidiative lipidomics coming of age:advances in analysis of oxidized phospholipids in physiology and pathology

Significance: Oxidized phospholipids are now well-recognized as markers of biological oxidative stress and bioactive molecules with both pro-inflammatory and anti-inflammatory effects. While analytical methods continue to be developed for studies of generic lipid oxidation, mass spectrometry (MS) has underpinned the advances in knowledge of specific oxidized phospholipids by allowing their identification and characterization, and is responsible for the expansion of oxidative lipidomics. Recent Advances: Studies of oxidized phospholipids in biological samples, both from animal models and clinical samples, have been facilitated by the recent improvements in MS, especially targeted routines that depend on the fragmentation pattern of the parent molecular ion and improved resolution and mass accuracy. MS can be used to identify selectively individual compounds or groups of compounds with common features, which greatly improves the sensitivity and specificity of detection. Application of these methods have enabled important advances in understanding the mechanisms of inflammatory diseases such as atherosclerosis, steatohepatitis, leprosy and cystic fibrosis, and offer potential for developing biomarkers of molecular aspects of the diseases. Critical Issues and Future Directions: The future in this field will depend on development of improved MS technologies, such as ion mobility, novel enrichment methods and databases and software for data analysis, owing to the very large amount of data generated in these experiments. Imaging of oxidized phospholipids in tissue MS is an additional exciting direction emerging that can be expected to advance understanding of physiology and disease

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

The process of lipid oxidation generates a diverse array of small aldehydes and carbonyl-containing compounds, which may occur in free form or esterified within phospholipids and cholesterol esters. These aldehydes mostly result from fragmentation of fatty acyl chains following radical oxidation, and the products can be subdivided into alkanals, alkenals (usually α,β-unsaturated), γ-substituted alkenals and bis-aldehydes. Isolevuglandins are non-fragmented di-carbonyl compounds derived from H2-isoprostanes, and oxidation of the ω−3-fatty acid docosahexenoic acid yield analogous 22 carbon neuroketals. Non-radical oxidation by hypochlorous acid can generate α-chlorofatty aldehydes from plasmenyl phospholipids. Most of these compounds are reactive and have generally been considered as toxic products of a deleterious process. The reactivity is especially high for the α,β-unsaturated alkenals, such as acrolein and crotonaldehyde, and for γ-substituted alkenals, of which 4-hydroxy-2-nonenal and 4-oxo-2-nonenal are best known. Nevertheless, in recent years several previously neglected aldehydes have been investigated and also found to have significant reactivity and biological effects; notable examples are 4-hydroxy-2-hexenal and 4-hydroxy-dodecadienal. This has led to substantial interest in the biological effects of all of these lipid oxidation products and their roles in disease, including proposals that HNE is a second messenger or signalling molecule. However, it is becoming clear that many of the effects elicited by these compounds relate to their propensity for forming adducts with nucleophilic groups on proteins, DNA and specific phospholipids. This emphasizes the need for good analytical methods, not just for free lipid oxidation products but also for the resulting adducts with biomolecules. The most informative methods are those utilizing HPLC separations and mass spectrometry, although analysis of the wide variety of possible adducts is very challenging. Nevertheless, evidence for the occurrence of lipid-derived aldehyde adducts in biological and clinical samples is building, and offers an exciting area of future research

Development of a low-maintenance measurement approach to continuously estimate methane emissions: a case study

The chemical breakdown of organic matter in landfills represents a significant source of methane gas (CH4). Current estimates suggest that landfills are responsible for between 3% and 19% of global anthropogenic emissions. The net CH4 emissions resulting from biogeochemical processes and their modulation by microbes in landfills are poorly constrained by imprecise knowledge of environmental constraints. The uncertainty in absolute CH4 emissions from landfills is therefore considerable. This study investigates a new method to estimate the temporal variability of CH4 emissions using meteorological and CH4 concentration measurements downwind of a landfill site in Suffolk, UK from July to September 2014, taking advantage of the statistics that such a measurement approach offers versus shorter-term, but more complex and instantaneously accurate, flux snapshots. Methane emissions were calculated from CH4 concentrations measured 700 m from the perimeter of the landfill with observed concentrations ranging from background to 46.4 ppm. Using an atmospheric dispersion model, we estimate a mean emission flux of 709 μg m−2 s−1 over this period, with a maximum value of 6.21 mg m−2 s−1, reflecting the wide natural variability in biogeochemical and other environmental controls on net site emission. The emissions calculated suggest that meteorological conditions have an influence on the magnitude of CH4 emissions. We also investigate the factors responsible for the large variability observed in the estimated CH4 emissions, and suggest that the largest component arises from uncertainty in the spatial distribution of CH4 emissions within the landfill area. The results determined using the low-maintenance approach discussed in this paper suggest that a network of cheaper, less precise CH4 sensors could be used to measure a continuous CH4 emission time series from a landfill site, something that is not practical using far-field approaches such as tracer release methods. Even though there are limitations to the approach described here, this easy, low-maintenance, low-cost method could be used by landfill operators to estimate time-averaged CH4 emissions and their impact downwind by simultaneously monitoring plume advection and CH4 concentrations

PARP1 Co-regulates EP300–BRG1-dependent transcription of genes involved in breast cancer cell proliferation and DNA repair

BRG1, an active subunit of the SWI/SNF chromatin-remodeling complex, enables the EP300-dependent transcription of proliferation and DNA repair genes from their E2F/CpG-driven promoters in breast cancer cells. In the current study, we show that BRG1–EP300 complexes are accompanied by poly-ADP-ribose polymerase 1 (PARP1), which emerges as the functional component of the promoter-bound multiprotein units that are capable of controlling gene expression. This enzyme is co-distributed with BRG1 at highly acetylated promoters of genes such as CDK4, LIG1, or NEIL3, which are responsible for cancer cell growth and the removal of DNA damage. ADP-ribosylation is necessary to maintain active transcription, since it ensures an open chromatin structure that allows high acetylation and low histone density. PARP1-mediated modification of BRG1 and EP300 does not affect the association of enzymes with gene promoters; however, it does activate EP300, which acetylates nucleosomes, leading to their eviction by BRG1, thus allowing mRNA synthesis. Although PARP1 was found at BRG1 positive/H3K27ac negative promoters of highly expressed genes in a transformed breast cancer cell line, its transcriptional activity was limited to genes simultaneously controlled by BRG1 and EP300, indicating that the ADP-ribosylation of EP300 plays a dominant role in the regulation of BRG1–EP300-driven transcription. In conclusion, PARP1 directs the transcription of some proliferation and DNA repair genes in breast cancer cells by the ADP-ribosylation of EP300, thereby causing its activation and marking nucleosomes for displacement by BRG1. PARP1 in rapidly dividing cells facilitates the expression of genes that confer a cancer cell phenotype. Our study shows a new mechanism that links PARP1 with the removal of DNA damage in breast cancer cells via the regulation of BRG1–EP300-dependent transcription of genes involved in DNA repair pathways

The effect of HOCl-induced modifications on phosphatase and tensin homolog (PTEN) structure and function

Oxidation by reactive species can cause changes in protein function and affect cell signaling pathways. Phosphatase and tensin homolog (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidized; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilization and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein

A mass spectrometry approach for the identification and localization of small aldehyde modifications of proteins

Lipids containing polyunsaturated fatty acids are primary targets of oxidation, which produces reactive short-chain aldehydes that can covalently modify proteins, a process called lipoxidation. Improved mass spectrometry (MS) methods for the analysis of these adducts in complex biological systems are needed. Lysozyme and human serum albumin (HSA) were used as model proteins to investigate lipoxidation products formed by two short-chain aldehydes, acrolein and pentanal, which are unsaturated and saturated aldehydes respectively. The adducts formed were stabilized by NaBH4 or NaBH3CN reduction and analysed by MS. Analysis of intact modified lysozyme showed a pentanal modification resulting from Schiff's base formation (+70 Da), and up to 8 acrolein adducts, all resulting from Michael addition (+58 Da). Analysis of tryptic digests identified specific histidine, cysteine and lysine residues modified in both lysozyme and HSA, and determined characteristic amino acid-specific fragmentations. Eight different internal fragment ions were found that could be used as general diagnostic ions for pentanal- and acrolein-modified amino acids. The combined use of intact protein analysis and LC-MS/MS methods provided a powerful tool for the identification and localization of aldehyde-protein adduct, and the diagnostic ions will facilitate the development of targeted MS methods for analysis of adducts in more complex samples

The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells

Background: The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid. Results: The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes. Conclusions: Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data

Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells

Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residues in vitro and in vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observed in vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity