Temporal dissociation of COX-2-dependent arachidonic acid and 2-arachidonoylglycerol metabolism in RAW264.7 macrophages.

Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation.

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown.

Electrostatic encoding of genome organization principles within single native nucleosomes.

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability.

Molecular Coping Mechanisms: Reprogramming tRNAs To Regulate Codon-Biased Translation of Stress Response Proteins.

As part of the classic central dogma of molecular biology, transfer RNAs (tRNAs) are integral to protein translation as the adaptor molecules that link the genetic code in messenger RNA (mRNA) to the amino acids in the growing peptide chain. tRNA function is complicated by the existence of 61 codons to specify 20 amino acids, with most amino acids coded by two or more synonymous codons. Further, there are often fewer tRNAs with unique anticodons than there are synonymous codons for an amino acid, with a single anticodon able to decode several codons by "wobbling" of the base pairs arising between the third base of the codon and the first position on the anticodon.

TGM2-mediated histone transglutamination is dictated by steric accessibility.

Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells.

Oncohistones: Exposing the nuances and vulnerabilities of epigenetic regulation.

Work over the last decade has uncovered a new layer of epigenetic dysregulation. It is now appreciated that somatic missense mutations in histones, the packaging agents of genomic DNA, are often associated with human pathologies, especially cancer. Although some of these "oncohistone" mutations are thought to be key drivers of cancer, the impacts of the majority of them on disease onset and progression remain to be elucidated.

A Genetically Encoded Approach for Breaking Chromatin Symmetry.

Nucleosomes frequently exist as asymmetric species in native chromatin contexts. Current methods for the traceless generation of these heterotypic chromatin substrates are inefficient and/or difficult to implement. Here, we report an application of the SpyCatcher/SpyTag system as a convenient route to assemble desymmetrized nucleoprotein complexes.

Janus Bioparticles: Asymmetric Nucleosomes and Their Preparation Using Chemical Biology Approaches.

The fundamental repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around two copies each of four canonical histone proteins. Nucleosomes possess 2-fold pseudo-symmetry that is subject to disruption in cellular contexts. For example, the post-translational modification (PTM) of histones plays an essential role in epigenetic regulation, and the introduction of a PTM on only one of the two "sister" histone copies in a given nucleosome eliminates the inherent symmetry of the complex.

Oncohistone mutations enhance chromatin remodeling and alter cell fates.

Whole-genome sequencing data mining efforts have revealed numerous histone mutations in a wide range of cancer types. These occur in all four core histones in both the tail and globular domains and remain largely uncharacterized. Here we used two high-throughput approaches, a DNA-barcoded mononucleosome library and a humanized yeast library, to profile the biochemical and cellular effects of these mutations.

Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks.

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO).

Detection of Cyclooxygenase-2-Derived Oxygenation Products of the Endogenous Cannabinoid 2-Arachidonoylglycerol in Mouse Brain.

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2-derived 2-AG oxygenation products has not been previously reported in neuronal tissue.

Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA.

Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50-100-fold higher in mtDNA than in nuclear DNA in several different human cell lines.

Protein Modification by Endogenously Generated Lipid Electrophiles: Mitochondria as the Source and Target.

Determining the impact of lipid electrophile-mediated protein damage that occurs during oxidative stress requires a comprehensive analysis of electrophile targets adducted under pathophysiological conditions. Incorporation of ω-alkynyl linoleic acid into the phospholipids of macrophages prior to activation by Kdo-lipid A, followed by protein extraction, click chemistry, and streptavidin affinity capture, enabled a systems-level survey of proteins adducted by lipid electrophiles generated endogenously during the inflammatory response. Results revealed a dramatic enrichment for membrane and mitochondrial proteins as targets for adduction.

Quantitative Analysis and Discovery of Lysine and Arginine Modifications.

Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location.

Nuclear Oxidation of a Major Peroxidation DNA Adduct, M1dG, in the Genome.

Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system.

Competition and allostery govern substrate selectivity of cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present.