James J. Galligan

Susceptibility of L-FABP−/− mice to oxidative stress in early-stage alcoholic liver

Chronic ethanol consumption is a prominent cause of liver disease worldwide. Dysregulation of an important lipid uptake and trafficking gene, liver-fatty acid binding protein (L-FABP), may contribute to alterations in lipid homeostasis during early-stage alcoholic liver. We have reported the detrimental effects of ethanol on the expression of L-FABP and hypothesize this may deleteriously impact metabolic networks regulating fatty acids. Male wild-type (WT) and L-FABP−/− mice were fed a modified Lieber-DeCarli liquid diet for six weeks. To assess the response to chronic ethanol ingestion, standard biochemical indicators for alcoholic liver disease (ALD) and oxidative stress were measured. Ethanol ingestion resulted in attenuation of hepatic triglyceride accumulation and elevation of cholesterol in L-FABP−/− mice. Lipidomics analysis validated multiple alterations in hepatic lipids resulting from ethanol treatment. Increased immunohistochemical staining for the reactive aldehydes 4-hydroxynonenal and malondialdehyde were observed in WT mice ingesting ethanol; however, L-FABP−/− mice displayed prominent protein adducts in liver sections evaluated from pair-fed and ethanol-fed mice. Likewise, alterations in glutathione, thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyl content all indicated L-FABP−/− mice exhibit high sustained oxidative stress in the liver. These data establish that L-FABP is an indirect antioxidant protein essential for sequestering FFA and that its impairment could contribute to in the pathogenesis of ALD.

From the exposome to mechanistic understanding of chemical-induced adverse effects

The exposome encompasses an individuals exposure to exogenous chemicals, as well as endogenous chemicals that are produced or altered in response to external stressors. While the exposome concept has been established for human health, its principles can be extended to include broader ecological issues. The assessment of exposure is tightly interlinked with hazard assessment. Here, we explore if mechanistic understanding of the causal links between exposure and adverse effects on human health and the environment can be improved by integrating the exposome approach with the adverse outcome pathway (AOP) concept that structures and organizes the sequence of biological events from an initial molecular interaction of a chemical with a biological target to an adverse outcome. Complementing exposome research with the AOP concept may facilitate a mechanistic understanding of stress-induced adverse effects, examine the relative contributions from various components of the exposome, determine the primary risk drivers in complex mixtures, and promote an integrative assessment of chemical risks for both human and environmental health.

Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice.

Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4−/− mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4−/− mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4−/− mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4−/− mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4−/− PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an important role in protecting against carbonylation of mitochondrial proteins.

Accumulation of isolevuglandin-modified protein in normal and fibrotic lung.

Protein lysine modification by γ-ketoaldehyde isomers derived from arachidonic acid, termed isolevuglandins (IsoLGs), is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by IsoLGs are subject to genetic regulation and the identity of IsoLG-modified proteins remain unclear. Herein we show that Nrf2 and Nox2 are key regulators of IsoLG modification in pulmonary tissue and report on the identity of proteins analyzed by LC-MS following immunoaffinity purification of IsoLG-modified proteins. Gene ontology analysis revealed that proteins in numerous cellular pathways are susceptible to IsoLG modification. Although cells tolerate basal levels of modification, exceeding them induces apoptosis. We found prominent modification in a murine model of radiation-induced pulmonary fibrosis and in idiopathic pulmonary fibrosis, two diseases considered to be promoted by gene-regulated oxidant stress. Based on these results we hypothesize that IsoLG modification is a hitherto unrecognized sequelae that contributes to radiation-induced pulmonary injury and IPF.

Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal.

Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue–residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.

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

Abstract 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.

Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks

Significance Chromatin 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. 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). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.

Abstract 3050: Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury

The development of radiation induced lung injury (RiPI) is a major barrier that limits the dose that can be administered for controlling locally advanced lung cancer. Although progress has been made toward identifying the pathophysiological events responsible for RiPI, there is still a substantial gap in knowledge. It is well established that oxidative stress is central to the progression of RiPR. In addition, reoccurring cellular injury appears to be a critical event for promotion of radiation-induced fibrosis. Yet, the exact mechanism by which reactive oxygen species (ROS) promotes injury and the nature of the reoccurring injury remain to be fully elucidated. Isolevuglandins are generated by free radical peroxidation of phospholipid-esterified arachidonic acid. Isoketalation, defined as covalent adduction of isolevuglandins to the e-amino group of protein lysine residues, is emerging as a novel mechanism by which ROS contributes to the genesis of some diseases. Although isoketalation promotes proteotoxicity and cytotoxicity that can contribute to disease progression, there is currently a lack of knowledge concerning its role in RiPI, the identity of susceptible proteins or whether the process can be genetically regulated. We used a multifaceted approach that included wild type, Nrf2 and p47 null mice, confocal microscopy, and LC-MS/MS analysis of affinity purified proteins to address these questions. Mass spectrometry and gene ontology analysis identified several potential protein targets involved in cytoskeletal regulation, Wnt signaling, integrin signaling, chemokine and cytokine signaling and histone biology. Genetic evidence linking oxidant challenge to protein adduction was provided from the mouse knockout studies where it was shown that Nrf2 expression suppressed, while NADPH oxidase activity promoted isoketalation. We found that when isoketalation exceeded a critical level, cells underwent apoptosis. We identified the cell types in mouse lung that are susceptible to adduction. In a C57BL/6j murine model of radiation induced pulmonary injury we found that ionizing radiation increased the level of adduction concomitant with the development of a chronic apoptotic phenotype. We used human idiopathic pulmonary fibrotic (IPF) tissue as a surrogate for radiation-induced pulmonary fibrotic tissue, which is very hard to obtain. Human IPF and radiation-induced lung injury share a common phenotype that includes slow/chronic development and a prominent ROS component. We found an abundance of adducted protein in human IPF compared to control lung tissue, identified collagen 1α1 as one of the highly adducted proteins and show that adduction impairs collagen degradation by MMP1. In summary, these data support the hypothesis that radiation-induced oxidant stress promotes pulmonary injury, in part, by a hitherto, unrecognized mechanism: isoketalation. Supported in part by NIH/NHLBI Grant RO1HL112286. Citation Format: Stacy Mont, Sean S. Davies, L Jackson Roberts, Raymond L. Mernaugh, W Hayes McDonald, Brahm H. Segal, Jonathan A. Kropski, Timothy S. Blackwell, Konjeti R. Sekhar, James J. Galligan, Lawrence J. Marnett, Michael L. Freeman. Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3050.

Abstract A12: Alkylation of histones by 4-oxo-2-nonenal as a novel modification linked to oxidative stress

Sustained oxidative stress leads to the generation of toxic concentrations of the lipid aldehydes 4-hydroxy-2-nonenal (4-HNE) and 4-oxononenal (4-ONE), which are capable of covalently modifying the side-chains of Cys, His, and Lys residues. These protein modifications have been identified as a contributing factor in numerous disease states, including cancer, cardiovascular disease, neurodegeneration and diabetes. Here, we describe a novel class of histone modifications resulting from lipid electrophile adduction of His and Lys residues. Utilizing click chemistry with ω-alkynyl-4-ONE (a4-ONE), we have identified all-four core histones as targets for modification in RKO cells treated with physiologically relevant concentrations of electrophile. Isolation of chromatin from these cells reveals H2B as a highly susceptible target to modification by 4-ONE, while H2A, H3 and H4 show minimal reactivity. Interestingly, similar experiments with a4-HNE failed to identify any of the core histones as targets for modification. 4-HNE reacts heavily with Cys residues, which histones lack, whereas 4-ONE reacts primarily with the e-amine of Lys residues. A proteomic investigation of site-specific modifications in RKO cells treated with 4-ONE reveals H2B Lys117 as a major target for modification. The predominant stable species detected on H2BK117 results from a 1,2-addition and net acylation analogous to Lys acetylation. H2BK117 is a surface exposed Lys residing on the face of the nucleosome core particle. Treatment of RKO cells with physiologically relevant doses of 4-ONE results in the altered expression of 40 genes. Electrophile adduction of histones provides a previously unexplored link between oxidative stress and gene regulation. Citation Format: J J. Galligan, K L. Rose, W N. Beavers, C D. Aluise, S C. Shuck, L J. Marnett. Alkylation of histones by 4-oxo-2-nonenal as a novel modification linked to oxidative stress. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A12.