SignificanceChromatin comprises the approximately 3 billion bases in the human genome and histone proteins. Histone posttranslational modifications (PTMs) regulate chromatin dynamics and protein transcription to expand the genetic code. Herein we describe the existence of Lys and Arg modifications on histones derived from a glycolytic by-product, methylglyoxal (MGO). These PTMs are abundant modifications, present at similar levels as those of modifications known to modulate chromatin function and leading to altered gene transcription. Using CRISPR-Cas9, we show that the deglycase DJ-1 protects histones from adduction by MGO. These findings demonstrate the existence of a previously undetected histone modification and provide a link between cellular metabolism and the histone code.

AP endonuclease 1 (APE1) is a multi-faceted protein with essential roles in DNA repair and transcriptional regulation. APE1 (Ref-1) activates many transcription factors (TF), including AP-1 and NF-κB. While the mechanism of APE1 redox activity remains unknown, it may involve reduction of an oxidized Cys in the TF DNA-binding domain. Several small molecules inhibit APE1-mediated TF activation, including the quinone derivative E3330. It has been proposed some inhibitors bind near C65, a residue suggested to be important for TF activation, but the binding site has not been determined for any inhibitor. Remarkably, NMR and molecular docking studies here reveal E3330 binds in the DNA repair active site of APE1, far removed from C65. Accordingly, AP endonuclease activity is substantially inhibited by E3330 (100 μM), suggesting that E3330 may not selectively inhibit APE1 redox activity in cells, in contrast with previous proposals. A naphthoquinone analog of E3330, RN7-60, binds a site removed from both C65 and the repair active-site. While a detailed understanding of how these inhibitors work requires further studies into the mechanism of redox activity, our results do not support proposals that E3330 binds selectively (and slowly) to locally unfolded APE1, or that E3330 promotes formation of disulfide bonds in APE1. Rather, we suggest E3330 may suppress a conformational change needed for redox activity, disrupt productive APE1-TF binding, or block the proposed redox chaperone activity of APE1. Our results provide the first structural information for any APE1 redox inhibitor, and could facilitate development of improved inhibitors for research and perhaps clinical purposes.

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. Here, we report that M1dG levels in intact DNA were increased from basal levels of 1 adduct per 108 nucleotides to 2 adducts per 106 nucleotides following adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also found that M1dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M1dG (to ~5 adducts per 107 nucleotides), and that this oxidation correlated with a decline in M1dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M1dG (1 adduct per 106 nucleotides) and the endogenous formation of 6-oxo-M1dG. This is the first report of the production of 6-oxo-M1dG in genomic DNA in intact cells, and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis, and repair.

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. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/−) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.

A literature survey and theoretical calculations have been applied to explore bilateral symmetry in natural product systems. Molecular bilateral symmetry is defined to include C(2) (sigma plane or axis), C(s)(), and C(2)(v)() point groups in molecules. Natural products that possess chirality in the form of C(2)-axes or sigma planes of symmetry are present in higher proportions (69%) compared to molecules bearing achiral C(s)() or C(2)(v)() point groups (14% and 16%, respectively). Density functional theoretical and semiempirical calculations indicate that the dimers 3,3'-dibromo-5,5'-[N-(2-(3-bromo-4-hydroxyphenyl)ethyl)-2-hydroxyiminoacetamide]biphenyl-2,2'-diol (1), (S,S)-1,2-bis(2-amino-3H-imidazol-4-yl)-(R,R)-3,4-bis(1H-pyrrole-2-amido)cyclobutane (2), 2-oxo-dimethyl-1,3-bis(3,4-dibromobenzene-1,2-diol) (11), 1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (12), and bis(5-isopropyl-8-methylazulene)methane (13) evolve more energy per connecting bond than the corresponding trimers or tetramers would. This we propose is a guiding parameter that may adjust molecule growth. The corresponding trimers, tetramers, or higher oligomers of 1, 2, and 11-13 appear to represent "missing" compounds in nature. Natural products 1, 2, and 11-13, having 3-fold and higher levels of symmetry, would founder on the lack of a facile method of synthesis and on the prohibitively high-energy costs caused by steric crowding at their core.

This paper examines ring size patterns of natural product macrocycles. Evidence is presented that natural macrocycles containing 14-, 16-, and 18-membered rings are of frequent occurrence based on a data mining study. The results raise a question about the limited diversity of macrocycle ring sizes and the nature of the constraints that may cause them. The data suggest that the preference bears no relationship to the odd-even frequency in natural fatty acids. The trends reported here, along with those reported previously (Wessjohann et al. (2005) Mol Divers 9:171), may be generalized to better understand the possible structure preferences of natural macrocycles.

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. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation.

During the synthetic pursuit of guanosine (triG) and xanthosine (triX) tricyclic nucleosides analogues, an interesting side product was discovered. In an effort to uncover the mechanistic factors leading to this result, a series of reaction conditions were investigated. It was found that by varying the conditions, the appearance of the side product could be controlled. In addition, the yield of the desired products could be manipulated to afford either a 50:50 mix of both triG and triX, or a majority of one or the other. To demonstrate the broad utility of the method, it was also adapted to the synthesis of guanosine and xanthosine from 5-amino-1-β-D-ribofuranosyl-4-imidazolecarboxyamide (AICAR). The mechanistic details surrounding the synthetic efforts are reported herein.