Amino Acid Adducts Research Articles

Evaluation of organophosphorus pesticide tyrosine adducts for postmortem change by human serum albumin with liquid chromatography quadrupole Orbitrap mass spectrometry.

Organophosphorus pesticides (OPPs) having a phosphate ester moiety, such as malathion (MA) and methidathion (DMTP), are widely used and have been detected in many fatal cases of accidental exposure or suicide in Japan. In forensic toxicology, the accurate determination of blood OPP concentration is mandatory to prove death by OPP poisoning. However, fatal pesticide concentration in blood at autopsy varies depending on the circumstances surrounding the dead body. In this study, we found that 16 OPPs were degraded by human serum albumin (HSA) in a temperature-dependent fashion. The mechanism underlying MA, DMTP, azinphos-methyl, etrimfos, fenthion (MPP), pirimiphos-methyl, (E)-dimethylvinphos, (Z)-dimethylvinphos, vamidothion, edifenphos (EDDP), fosthiazate, and pyraclofos degradation involves the formation of adducts with tyrosine residues in HSA. The mass spectra obtained by liquid chromatography quadrupole Orbitrap mass spectrometry revealed that phosphate ester amino acid adducts such as Y-adduct1, Y-adduct2, Y-adduct3, Y-adduct4, and Y-adduct5 were formed in HSA solution incubated with OPPs. These results indicate that the 16 OPPs are postmortem changed by HSA. The detection of phosphate ester amino acid adducts such as Y-adduct1, Y-adduct2, Y-adduct3, Y-adduct4, and Y-adduct5, instead of MA, DMTP, azinphos-methyl, etrimfos, MPP, pirimiphos-methyl, (E)-dimethylvinphos, (Z)-dimethylvinphos, vamidothion, EDDP, fosthiazate, and pyraclofos per se, may be used to determine death by these OPPs poisoning.

Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-α. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking. We used single-cell cloning, generation of recombinant antibodies (n= 356 from 25 donors), and antigen-screening to investigate the presence of class-switched MDA/MAA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage. Anti-MDA/MAA+ B cells were found in bone marrow plasma cells of late disease and in the lung of both early disease and risk-individuals and in different B cell subsets (memory, double negative B cells). These were compared with previously identified anti-MDA/MAA from synovial memory and plasma cells. Seven out of eight clones carried somatic hypermutations and all bound MDA/MAA-lysine independently of protein backbone. However, clones with somatic hypermutations targeted MAA cross-linked structures rather than MDA- or MAA-hapten, while the germline-encoded synovial clone instead bound linear MDA-lysine in proteins and peptides. Binding patterns were maintained in germline converted clones. Affinity purification of polyclonal anti-MDA/MAA from patient serum revealed higher proportion of anti-MAA versus anti-MDA compared to healthy controls. In conclusion, IgG anti-MDA/MAA show distinct targeting of different molecular structures. Anti-MAA IgG has been shown to promote bone loss and osteoclastogenesis invivo and may contribute to RA pathogenesis.

Phenolic Acid-Amino Acid Adducts Exert Distinct Immunomodulatory Effects in Macrophages Compared to Parent Phenolic Acids.

Caffeic acid (CA) and chlorogenic acid (CGA) are commonly found phenolic acids in plant-derived foods and beverages. Their corresponding adducts with cysteine (Cys) have been detected in coffee-containing beverages. However, despite the well-documented antioxidant and anti-inflammatory activity of CA and CGA, the immunomodulatory activities of the Cys adducts (CA-Cys and CGA-Cys) are unknown. The adducts were therefore synthesized, and their immunomodulatory effects were studied in lipopolysaccharide (LPS)-treated RAW 264.7 cells and compared to the activity of the parent phenolic acids. CA and CGA generally down-regulated the inflammatory responses. However, RNA-sequencing showed that the LPS-induced pathways related to Toll-like receptor signaling, chemokine signaling, and NOD-like receptor signaling, and JAK-STAT/MAPK signaling pathways were upregulated in adduct-treated cells relative to parent phenolic acids, while neurodegenerative disorder-related pathways and metabolic pathways were downregulated. Production of prostaglandin E2 (PGE2), interleukin-6, tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) was all inhibited by CA and CGA (P < 0.05). PGE2 and TNF-α were further suppressed in adduct-stimulated cells (P < 0.05), but ROS production was increased. For example, TNF-α produced by 100 μM CGA-stimulated cells and 100 μM CGA-Cys adduct-stimulated cells were 4.46 ± 0.23 and 1.61 ± 0.18 ng/mL, respectively. Thus, the addition of the Cys moiety drastically alters the anti-inflammatory activity of phenolic compounds.

Simultaneous quantification of multiple amino acid adducts from sulfur mustard-modified human serum albumin in plasma at trace exposure levels by ultra-high performance liquid chromatography–triple quadrupole mass spectrometry after propionyl derivatization

Sulfur mustard (HD) is a highly toxic vesicant and is prohibited by the Organisation for the Prohibition of Chemical Weapons (OPCW). HD can modify human serum albumin (HSA) to generate hydroxyethylthioethyl (HETE) adducts, which could be utilized as biomarkers for verifying HD exposure in forensic analysis. Here, five amino acid adducts generated from pronase digestion of HD-exposed human serum albumin (HD–HSA) in plasma were selected as biomarkers to retrospectively detect HD exposure. HD–HSA was precipitated from plasma with acetone, digested by pronase, derivatized with propionic anhydride (PA), and analysed with ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC–TQ MS). The limits of detection (LODs) and limits of quantification (LOQs) of the HD exposure concentrations were evaluated as 1.00 ng/mL at S/N≥3 and 3.00 ng/mL at S/N≥10, respectively, which are approximately 60 times lower than those of the reported method. The approach shows good linearity (R2≥0.997) from 3.00 ng/mL to 10.0 µg/mL of HD-exposed human plasma with satisfactory precision and accuracy. The developed approach was applied to analysing samples from the 6th OPCW Biomedical Proficiency Test (BioPT). The study showed that the developed approach was also suitable for analysing human plasma samples that were exposed to six of HD analogues, which were common impurities in sulfur mustard mixtures. Moreover, the method was successfully applied to plasma from other species, including rabbits, rats and cattle. This study provides a reliable and sensitive tool for the retrospective detection of vesicants exposure based on multiple biomarkers.

Evaluation of Post-Mortem Interaction between Hemoglobin and Oxime-Type Carbamate Pesticides.

Oxime-type carbamate pesticides having an oxime moiety such as aldicarb, butocarboxim, methomyl, oxamyl, and thiofanox are widely used and have been detected in many fatal cases of accidental exposure or suicide. In forensic toxicology, the accurate determination of blood pesticide concentration is obligatory to prove death by oxime-type carbamate pesticide poisoning. However, the fatal pesticide concentration in blood at autopsy differs from that at the time of death. In this study, we found that oxime-type carbamate pesticides were decomposed by Hb in a temperature-dependent fashion. The mechanism underlying methomyl, aldicarb, oxamyl, and thiofanox decomposition involves the formation of adducts with the amino acids in Hb. With regard to butocarboxim, its decomposition involves the oxidation of the free form and the formation of adducts with the amino acids in Hb. The mass spectra obtained by liquid chromatography quadrupole time-of-flight mass spectrometry revealed that carbamylated amino acid adducts such as Wcar-adduct and Vcar-adduct were formed in Hb solution incubated with methomyl, aldicarb, oxamyl, and thiofanox, whereas alkylated amino acid adducts such as Walkyl-adduct were formed in Hb solution incubated with butocarboxim. These results indicate that aldicarb, butocarboxim, methomyl, oxamyl, and thiofanox are post-mortem changed by Hb.

Alkylated glutamic acid and histidine derived from protein-adducts indicate exposure to sulfur mustard in avian serum.

Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals. For legal reasons, bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins, for example, serum albumin (SA), are valuable long-lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck, and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant, and re-dissolution analysis were performed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry in the selected reaction monitoring mode, μLC-ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE]+ at m/z 105.0. After in vitro incubation with SM and pronase-catalyzed proteolysis, the alkylated amino acids Glu(-HETE) and His(-HETE) were detected. Both borne the SM-characteristic HETE-moiety bound to their side chain. The eightfold deuterated SM analog (d8-SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50°C), and time to maximize the yield of Glu(-HETE) (30 min) and His(-HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein-adducts were stable in serum at -30°C for at least 33 days and for three freeze-and-thaw cycles. At the body temperature of chicken (+40°C), Glu(-HETE) was degraded in serum (period of half-change 3 days), whereas His(-HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM.

Adducts of Lower Fullerenes and Amino Acids: Synthesis, Identification, and Quantum-Mechanical Modeling of Their Physicochemical Properties

Different ways of synthesizing bis-, tris-, and octakis-adducts of C60 and C70 lower fullerenes are considered, and their yield and purity are described. The adducts are identified by physicochemical means: elemental analysis, IR, electron spectroscopy, Raman spectroscopy, HPLC, mass spectrometry, and complex thermal analysis. Their physicochemical properties are modeled using computers, density functional theory, and molecular dynamics at the atomic-molecular level.

Site-specific protein modification by 3-n-butylphthalide in primary hepatocytes: Covalent protein adducts diminished by glutathione and N-acetylcysteine

Aims3-n-Butylphthalide (NBP) is widely used for the treatment of cerebral ischaemic stroke but can causeliver injury in clinical practice. This study aims to elucidate the underlying mechanisms and propose potential preventive strategies. Main methodsNBP and its four major metabolites, 3-hydroxy-NBP (3-OH-NBP), 10-hydroxy-NBP, 10-keto-NBP and NBP-11-oic acid, were synthesized and evaluated in primary human or rat hepatocytes (PHHs, PRHs). NBP-related substances or amino acid adducts were identified and semi-quantitated by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The target proteins and binding sites were identified by shotgun proteomics based on peptide mass fingerprinting coupled with tandem mass spectrometry and verified by molecular docking. Key findingsThe toxicity of NBP and its four major metabolites were compared in both PHHs and PRHs, and 3-OH-NBP was found to be the most toxic metabolite. 3-OH-NBP induced remarkable cell death and oxidative stresses in hepatocytes, which correlated well with the levels of glutathione and N-acetylcysteine adducts (3-GSH-NBP and 3-NAC-NBP) in cell supernatants. Additionally, 3-OH-NBP covalently conjugated with intracellular Cys, Lys and Ser, with preferable binding to Cys sites at Myh9 Cys1380, Prdx4 Cys53, Vdac2 Cys48 and Vdac3 Cys36. Furthermore, we found that CYP3A4 induction by rifampicin augmented NBP-induced cell toxicity and supplementing with GSH or NAC alleviated the oxidative stresses and reactive metabolites caused by 3-OH-NBP. SignificanceOur work suggests that glutathione depletion, mitochondrial injury and covalent protein modification are the main causes of NBP-induced hepatotoxicity, which may be prevented by exogenous GSH or NAC supplementation and avoiding concomitant use of CYP3A4 inducers.

Physicochemical Properties of Adducts of Light Fullerenes and Amino Acids

Physicochemical Properties of Adducts of Light Fullerenes and Amino Acids

Developing urinary pyrrole-amino acid adducts as non-invasive biomarkers for identifying pyrrolizidine alkaloids-induced liver injury in human.

Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Currently, a definitive diagnostic method for PA-induced liver injury (PA-ILI) is lacking. In the present study, using a newly developed analytical method, we identified four pyrrole-amino acid adducts (PAAAs), namely pyrrole-7-cysteine, pyrrole-9-cysteine, pyrrole-9-histidine, and pyrrole-7-acetylcysteine, which are generated from reactive pyrrolic metabolites of PAs, in the urine of PA-treated male Sprague Dawley rats and PA-ILI patients. The elimination profiles, abundance, and persistence of PAAAs were systematically investigated first in PA-treated rat models via oral administration of retrorsine at a single dose of 40 mg/kg and multiple doses of 5 mg/kg/day for 14 consecutive days, confirming that these urinary excreted PAAAs were derived specifically from PA exposure. Moreover, we determined that these PAAAs were detected in ~ 82% (129/158) of urine samples collected from ~ 91% (58/64) of PA-ILI patients with pyrrole-7-cysteine and pyrrole-9-histidine detectable in urine samples collected at 3 months or longer times after hospital admission, indicating adequate persistence time for use as a clinical test. As direct evidence of PA exposure, we propose that PAAAs can be used as a biomarker of PA exposure and the measurement of urinary PAAAs could be used as a non-invasive test assisting the definitive diagnosis of PA-ILI in patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00204-021-03129-6.

Nuclear Sphingosine-1-phosphate Lyase Generated ∆2-hexadecenal is A Regulator of HDAC Activity and Chromatin Remodeling in Lung Epithelial Cells.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to ∆2-hexadecenal (∆2-HDE) and ethanolamine phosphate by S1P lyase (S1PL) in mammalian cells. We have recently demonstrated the activation of nuclear SPHK2 and the generation of S1P in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa. Here, we have investigated the nuclear localization of S1PL and the role of ∆2-HDE generated from S1P in the nucleus as a modulator of histone deacetylase (HDAC) activity and histone acetylation. Electron micrographs of the nuclear fractions isolated from MLE-12 cells showed nuclei free of ER contamination, and S1PL activity was detected in nuclear fractions isolated from primary lung bronchial epithelial cells and alveolar epithelial MLE-12 cells. Pseudomonas aeruginosa-mediated nuclear ∆2-HDE generation, and H3/H4 histone acetylation was attenuated by S1PL inhibitors in MLE-12 cells and human bronchial epithelial cells. In vitro, the addition of exogenous ∆2-HDE (100-10,000 nM) to lung epithelial cell nuclear preparations inhibited HDAC1/2 activity, and increased acetylation of Histone H3 and H4, whereas similar concentrations of S1P did not show a significant change. In addition, incubation of ∆2-HDE with rHDAC1 generated five different amino acid adducts as detected by LC-MS/MS; the predominant adduct being ∆2-HDE with lysine residues of HDAC1. Together, these data show an important role for the nuclear S1PL-derived ∆2-HDE in the modification of HDAC activity, histone acetylation, and chromatin remodeling in lung epithelial cells.

Investigations on the formation of α-dicarbonyl compounds and 5-hydroxymethylfurfural in fruit products during storage: New insights into the role of Maillard reaction

The formation of α-dicarbonyl compounds and 5-hydroxymethylfurfural was investigated under different conditions and the amino acid adducts of them were confirmed using high-resolution mass spectrometry in fruit products during storage. Changes in the concentrations of sugars, amino acids, α-dicarbonyl compounds, and 5-hydroxymethylfurfural in fruit juice concentrates and dried fruits were monitored. Among the dicarbonyls, glucosone was the dominant one in 30 °Bx of fruit juice concentrates, whereas 3-deoxyglucosone was the major in 50 and 70 °Bx of those and in all dried fruits during storage. The highest level of 3-deoxyglucosone was found as 7251 ± 896.6 mg/kg in dried date at the end of the storage. During storage, the loss of free amino acids significantly increased (p < 0.05) in the higher initial reactant concentrations in fruit juice concentrates. The confirmation of amino acid adducts of dicarbonyls and 5-hydroxymethylfurfural generally with high mass accuracy proved the contribution of Maillard reaction to non-enzymatic reactions in fruit products.

A Workflow towards the Reproducible Identification and Quantitation of Protein Carbonylation Sites in Human Plasma.

Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA.

Physicochemical properties, biological activity and biocompatibility of water-soluble C60-Hyp adduct

Amino acid adducts of light fullerenes have a potential of application in a variety of fields of biomedicine, that is reactive oxygen species scavenging activity, anticancer activity, viruses and bacteria inactivation etc. In this work, the water-soluble C60 fullerene derivative with l-hydroxyproline (C60(C5H9NO3)2, C60-Hyp) was studied. Extensive biomedical investigation of this compound, namely, antiradical activity in the reaction with stable diphenylpicrylhydrazyl radical, the binding to human serum albumin, photodynamic properties, cytotoxicity in glioblastoma A172 and lung carcinoma A549 cell lines, erythrocytes haemolysis, platelet aggregation, genotoxicity on human peripheral blood mononuclear cells was conducted. Moreover, the dynamic and structural characteristics of C60-Hyp–H2O binary system were obtained using molecular dynamic (MD) method, and size distribution along with ζ-potentials of C60-Hyp associates was measured.

Toxicometabolomics of the new psychoactive substances \u03b1-PBP and \u03b1-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts

Toxicometabolomics, essentially applying metabolomics to toxicology of endogenous compounds such as drugs of abuse or new psychoactive substances (NPS), can be investigated by using different in vitro models and dedicated metabolomics techniques to enhance the number of relevant findings. The present study aimed to study the toxicometabolomics of the two NPS α-pyrrolidinobutiophenone (1-phenyl-2-(pyrrolidin-1-yl)butan-1-one, α-PBP) and α-pyrrolidinoheptaphenone (1-phenyl-2-(pyrrolidin-1-yl)heptan-1-one, α-PEP, PV8) in HepaRG cell line incubates. Evaluation was performed using reversed-phase and normal-phase liquid chromatography coupled with high-resolution mass spectrometry in positive and negative ionization mode, respectively, to analyze cells and cell media. Statistical evaluation was performed using one-way ANOVA, principal component discriminant function analysis, as well as hierarchical clustering. In general, the analysis of cells did not mainly reveal any features, but the parent compounds of the drugs of abuse. For α-PBP an increase in N-methylnicotinamide was found, which may indicate hepatotoxic potential of the substance. After analysis of cell media, significant features led to the identification of several metabolites of both compounds. Amino acid adducts with glycine and alanine were found, and these have not been described in any study before and are likely to appear in vivo. Additionally, significant changes in the metabolism of cholesterol were revealed after incubation with α-PEP. In summary, the application of metabolomics techniques after HepaRG cells exposure to NPS did not lead to an increased number of identified drug metabolites compared to previously published studies, but gave a wider perspective on the physiological effect of the investigated compounds on human liver cells.

Separation of 13C- and 15N-Isotopologues of Amino Acids with a Primary Amine without Mass Resolution by Means of O-Phthalaldehyde Derivatization and Collision Induced Dissociation.

Computational and experimental advances of recent years have culminated in establishing 13C-Metabolic Flux Analysis (13C-MFA) as a routine methodology to unravel the fluxome. As the acronym suggests, 13C-MFA has relied on the relative abundance of 13C-isotopes in metabolites for flux inference, most commonly measured by mass spectrometry. In this manuscript we expand the scope of labeling measurements to the case of simultaneous 13C- and 15N-labeling of amino acids. Analytically, the separation of isotopologues of this metabolite class can only be achieved at resolving power beyond 65,000. In this manuscript we harvest an overlooked property of the collision induced dissociation of amino acid adducts to discern 13C- and 15N- isotopologues of amino acids with a primary amine without separating them in the m/z domain.

Bioactivation of GPR40 Agonist MK-8666: Formation of Protein Adducts in Vitro from Reactive Acyl Glucuronide and Acyl CoA Thioester.

MK-8666, a selective GPR40 agonist developed for the treatment of type 2 diabetes mellitus, was discontinued in phase I clinical trials due to liver safety concerns. To address whether chemically reactive metabolites played a causative role in the observed drug induced liver injury (DILI), we characterized the metabolism, covalent binding to proteins, and amino acid targets of MK-8666 in rat and human hepatocytes or cofactor-fortified liver microsomes. MK-8666 was primarily metabolized to an acyl glucuronide in hepatocytes of both species and a taurine conjugate in rat hepatocytes. Similar levels of covalent binding to proteins were observed in rat and human hepatocytes following incubation with [3H]MK-8666. After protease digestion of hepatocyte pellets, amino acid adducts A1, A2, and A3 were identified as transacylated products with lysine, serine, and cysteine residues, respectively. Amino acid adducts A4a-c were identified as glycation adducts resulting from rearrangement of MK-8666-1-O-β-acyl glucuronide to ring-opened aldehydes which further condensed with lysine residues of proteins into imine adducts. Adducts A1-A3 and A4a-c were detected in rat and human liver microsomes fortified with UDPGA. Adducts A1-A3 were detected in rat and human liver microsomes fortified with CoA and ATP. Additionally, a trace amount of CoA thioester metabolite of MK-8666 and its transacylated GSH adduct were detected in human liver microsomes fortified with CoA, ATP, and GSH. Higher levels of covalent binding to protein were observed when [3H]MK-8666 was incubated in liver microsomes supplemented with CoA and ATP compared to UDPGA. Addition of GSH attenuated levels of CoA thioester-mediated covalent binding by 41-45%. Collectively, these studies indicated that metabolism of the -COOH moiety of MK-8666 can form a reactive acyl glucuronide and an acyl CoA thioester, which covalently modifies proteins and may represent one causative mechanism of the observed DILI.

Correction to: Towards lignin-protein crosslinking: amino acid adducts of a lignin model quinone methide

The manuscript initially failed to cite the work of Dr. Cong et al., which was the basis for this effort. We apologize for this error.

Non-covalently bonded diastereomeric adducts of amino acids and (S)-1-phenylethanol in low-energy dissociative collisions

We have studied the collision induced dissociation reactions of proton-bound diastereomeric adducts of S-1-phenylethanol and enantiomers of three different amino acids (tryptophan, phenylalanine, methionine). In all cases, the loss of S-1-phenylethanol from the adduct ion is the only observed process, and the relative abundance is found to be independent of the chirality of the amino acid. This is in contrast to earlier experiments on the dissociation of protonated tryptophan–2-butanol adducts, where chirality affected the results. Results obtained from quantum chemical computations support and provide a rationale for the experimental observations and highlight temperature as a possible factor of importance for the chiral effect in these types of systems.

Δ‐2 Hexadecenal Generated from S1P by Nuclear S1P Lyase Is a Regulator of HDAC1/2 Activity and Histone Acetylation in Lung Epithelial Cells

Sphingosine‐1‐phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to Δ‐2 hexadecenal (Δ2HD) and ethanolamine‐phosphate by S1P lyase (S1PL) in mammalian cells. While the role of S1P as a key regulator of cellular functions in normal and pathological conditions is well established, the physiological role of fatty aldehyde released from S1P by S1PL is still unclear. We have recently demonstrated activation of nuclear SPHK2 and generation of S1P and Δ‐2 Hexadecenal in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa (PA). Here, we have investigated the nuclear localization of S1PL and the role of Δ2HD as a modulator of nuclear HDAC activity and histone acetylation.Nuclear extracts, free from cytosolic and ER contamination, prepared by detergent fractionation method, were probed for purity by Western blotting using organelle‐specific markers Lamin B, ERp72, and GAPDH. Nuclear fractions derived from primary lung bronchial epithelial cells and alveolar epithelial MLE‐12 cell line exhibited S1PL activity, as measured by a fluorogenic substrate assay and mass spectrometric analysis and immunostained for S1PL by Western blotting. Activation of lung epithelial cells with heat‐inactivated Pseudomonas aeruginosa (PA) stimulated Δ2HD generation in the nucleus, which was S1PL dependent. Blocking S1PL activity with an inhibitor, compound C1 (5μM), in MLE‐12 cells inhibited PA‐induced nuclear Δ2HD production and H3 and H4 histone acetylation. In vitro, addition of exogenous Δ2HD (100–10000 nM) to lung epithelial cell nuclear preparation inhibited HDAC 1/2 activity, and increased acetylation of Histone H3 and H4. Overexpressing wild‐type Sgpl1 gene into mouse embryonic fibroblasts (MEFs) deficient in Sgpl1 decreased basal and PA‐induced nuclear HDAC activity, underlining the role of S1P degradative products in modulating HDAC activity. Furthermore, in Sgpl1 −/−MEFs, PA‐induced HDAC activity was higher compared to Sgpl1+/+MEFs treated with PA. Additionally, incubation of Δ2HD with HDAC1 generated 5 different amino acid adducts as detected by LC‐MS/MS; the predominant adduct being Δ2HD with lysine residues of HDAC1.These data show nuclear localization of S1PL in lung epithelial cells and indicate a crucial epigenetic role for Δ2HD, released from S1P by S1PL in the nucleus, in regulating HDAC1/2 activity and histone acetylation. Thus, nuclear S1PL and generation Δ2HD may have a physiological and pathophysiological relevance to lung inflammatory injury.Support or Funding InformationThis work was supported by NIH grant HL 098050 to VN.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.