A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids.
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ABSTRACT:
SUBMITTER: Byzia A
PROVIDER: S-EPMC3984412 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In chronic inflammatory lung disorders such as chronic obstructive pulmonary disease (COPD), the concurrent organ-specific and systemic inflammatory responses lead to airway remodelling and vascular dysfunction. Although a major common risk factor for COPD, cigarette smoke alone cannot explain the progression of this disease; there is increasing evidence that genetic predisposition also plays a role in COPD susceptibility and progression. A key enzyme in chronic lung inflammation is leukotriene A4 hydrolase (LTA4H). With its aminopeptidase activity, LTA4H degrades the neutrophil chemoattractant tripeptide PGP. In this study, we used the luciferase reporter gene analysis system and quantitative trait locus analysis to explore the impact of single-nucleotide polymorphisms (SNPs) in the putative promoter region of LTA4H on LTA4H expression. We show that not only is the putative promoter of LTA4H larger than previously reported but also that SNPs in the expanded promoter region regulate expression of LTA4H both in cell-based systems and in peripheral blood samples from human subjects. These findings provide significant evidence for an active region upstream of the previously reported LTA4H promoter, which may have implications related to ongoing inflammatory processes in chronic lung disease.
| S-EPMC5005155 | biostudies-literature
Project description:Asthma is associated with the overproduction of leukotrienes (LTs), including LTB4. Patients with severe asthma can be highly responsive to 5-lipoxygenase (5-LO) inhibition, which blocks production of both the cysteinyl LTs and LTB4. Production of LTB4 has traditionally been ascribed to neutrophils, mononuclear phagocytes, and epithelial cells, and acts as a chemoattractant for inflammatory cells associated with asthma. The source of LTB4 is unclear, especially in eosinophilic asthma. We speculated that the benefit of 5-LO inhibition could be mediated in part by inhibition of eosinophil-derived LTB4. LTB4 concentrations were assayed in BAL fluid from patients with severe asthma characterized by isolated neutrophilic, eosinophilic, and paucigranulocytic inflammation. Expression of LTA4 hydrolase (LTA4H) by airway eosinophils was determined by immunohistochemistry (IHC). Subsequently, peripheral blood eosinophils were activated and secreted LTB4 was quantified by enzyme immunoassay. Blood eosinophil LTA4H expression was determined by flow cytometry, qPCR, and IHC. LTB4 concentrations were elevated in BAL fluid from patients with severe asthma, including those with isolated eosinophilic inflammation, and these eosinophils displayed LTA4H via IHC. LTA4H expression by blood eosinophils was confirmed by flow cytometry, IHC, and qPCR. Robust LTB4 production by blood eosinophils was observed in response to some, but not all, stimuli. We demonstrated that eosinophils express LTA4H transcripts and protein, and can be stimulated to secrete LTB4. We speculate that in many patients with asthma, eosinophil-derived LTB4 is increased, and this may contribute to the efficacy of 5-LO inhibition.
| S-EPMC6444630 | biostudies-literature
Project description:Maresins are produced by macrophages from docosahexaenoic acid (DHA) and exert potent proresolving and tissue homeostatic actions. Maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid) is the first identified maresin. Here, we investigate formation, stereochemistry, and precursor role of 13,14-epoxy-docosahexaenoic acid, an intermediate in MaR1 biosynthesis. The 14-lipoxygenation of DHA by human macrophage 12-lipoxygenase (hm12-LOX) gave 14-hydro(peroxy)-docosahexaenoic acid (14-HpDHA), as well as several dihydroxy-docosahexaenoic acids, implicating an epoxide intermediate formation by this enzyme. Using a stereo-controlled synthesis, enantiomerically pure 13S,14S-epoxy-docosa-4Z,7Z,9E,11E,16Z,19Z-hexaenoic acid (13S,14S-epoxy-DHA) was prepared, and its stereochemistry was confirmed by NMR spectroscopy. When this 13S,14S-epoxide was incubated with human macrophages, it was converted to MaR1. The synthetic 13S,14S-epoxide inhibited leukotriene B4 (LTB4) formation by human leukotriene A4 hydrolase (LTA4H) ?40% (P<0.05) to a similar extent as LTA4 (?50%, P<0.05) but was not converted to MaR1 by this enzyme. 13S,14S-epoxy-DHA also reduced (?60%; P<0.05) arachidonic acid conversion by hm12-LOX and promoted conversion of M1 macrophages to M2 phenotype, which produced more MaR1 from the epoxide than M1. Together, these findings establish the biosynthesis of the 13S,14S-epoxide, its absolute stereochemistry, its precursor role in MaR1 biosynthesis, and its own intrinsic bioactivity. Given its actions and role in MaR1 biosynthesis, this epoxide is now termed 13,14-epoxy-maresin (13,14-eMaR) and exhibits new mechanisms in resolution of inflammation in its ability to inhibit proinflammatory mediator production by LTA4 hydrolase and to block arachidonate conversion by human 12-LOX rather than merely terminating phagocyte involvement.
| S-EPMC3688739 | biostudies-literature
Project description:ObjectiveAtherosclerosis is an inflammatory process resulting from the interaction between genetic and environmental factors. Leukotrienes are inflammatory mediators generated from arachidonic acid, and genetic polymorphisms involved in leukotriene metabolism are implicated in atherosclerosis. The objectives of this study are to examine whether genetic variants in key leukotriene enzymes are associated with atherosclerosis, and whether dietary intake of competing leukotriene substrates modifies the effect of leukotriene variants on atherosclerosis.MethodsAtherosclerosis was assessed by common carotid intima-media thickness (IMT) using ultrasound. Sequence variants within arachidonate 5-lipoxygenase activating protein (ALOX5AP) and leukotriene A4 hydrolase (LTA4H) genes were analyzed with 32 single nucleotide polymorphisms (SNPs) in 169 Caucasian twin pairs from the Vietnam Era Twin Registry. The associations between genetic polymorphisms and carotid atherosclerosis, and gene × diet interactions were examined by generalized estimating equation controlling for potential confounders.ResultsA six-SNP haplotype in LTA4H, designated HapE, was significantly associated with carotid IMT after adjusting for known coronary risk factors. Twins carrying HapE had a much lower IMT compared to twins not carrying (695 μm vs. 750 μm, p = 0.0007). Moreover, dietary intake of polyunsaturated fatty acids strongly augmented the cardioprotective effect of HapE among those with this haplotype but not those without, suggesting a haplotype × diet interaction (interaction P(HapE×n-3) = 0.03, P(HapE×n-6) = 0.015).ConclusionWe identified a novel leukotriene haplotype that appears to be protective toward subclinical atherosclerosis. This association is modified by dietary intake of polyunsaturated fatty acids.
| S-EPMC3630507 | biostudies-literature
Project description:Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)/aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzyme's ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4.
| S-EPMC123628 | biostudies-literature
Project description:The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme with epoxy hydrolase and aminopeptidase activities. We hypothesize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)-induced emphysema by clearing proline-glycine-proline (PGP), a triamino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild-type mice to CS over 5 mo while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bioproduction of leukotriene B4. Emphysematous phenotypes were assessed by premortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the bronchoalveolar lavage fluid and infiltration of neutrophils into the lungs were significantly reduced without affecting the levels of leukotriene B4. This protected murine lungs from CS-induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the leukotriene A4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema.
| S-EPMC4083682 | biostudies-literature
Project description:We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein-protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.
| S-EPMC2722745 | biostudies-literature
Project description:OBJECTIVE:To assess whether genetic variants involved in inflammation play a role in the sex difference in depression. Depression is, in part, genetically determined and inflammation has been implicated. Women are twice as likely to develop depression as men. METHODS:We examined the association, separately in men and women, between seven single nucleotide polymorphisms (SNPs) in the arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene and 12 SNPs in the leukotriene A4 hydrolase (LTA4H) gene and depression in 1368 white subjects (30.4% female) referred for cardiovascular evaluation. Depression was defined as a score of >or=10 in the Patient Health Questionnaire 9. Single marker analysis was assessed by the chi(2) test. Haplotype-specific associations were performed, using likelihood ratio tests. Empirical significance levels were determined by permutation tests. RESULTS:Depressed individuals, comprising 14.5% of the total, were more likely to be female, current smokers, have a history of diabetes and myocardial infarction. None of the SNPs in the ALOX5AP gene, either singly or in combination, was associated with depression. The 12 SNPs in the LTA4H gene were not individually associated with depression. However, a six-SNP haplotype in LTA4H gene, named HapE, showed a significant protective effect on depression in women, but not in men, after correcting for cardiovascular effects. The interaction between HapE and sex on depression was statistically significant. CONCLUSION:This study provides the first evidence for a sex-specific association of a novel haplotype in the LTA4H gene on depression. Although replication is needed, our study suggests that genetic variations may underlie sex differences in depression.
| S-EPMC3113512 | biostudies-literature
Project description:Leukotriene A4 hydrolase (LTA4H) is a key enzyme in the eicosanoid pathway. lta4h locus polymorphisms have previously been linked to tuberculosis (TB) susceptibility and disease outcome in a Vietnamese dataset, but further studies suggested that those results were poorly reproducible. We, therefore, compared the full set of variants (113 SNPs) within the gene in a Portuguese dataset of 112 TB patients and 120 controls, using both the frequency of SNPs and haplotypes, in order to assess their association with TB susceptibility. Although we obtained no significant differences between the TB patients and the control group, linkage analysis showed that an extensively typed polymorphism, rs17525495, was associated with 21 other SNPs, all displaying evidence of association to lower LTA4H expression. While the derived alleles of these SNPs showed a moderately higher frequency in the TB group, differences were not significant. In contrast to Asian populations, where these SNPs are much more frequent, the low frequencies of candidate SNPs in Europeans render them less pertinent in a public health context. Consequently, the typing of specific polymorphisms as a strategy to establish preventive measures and differential TB drug treatments is important but needs to take into consideration that haplotypic background and structure can be substantially different in distinct geographic regions.
| S-EPMC6956305 | biostudies-literature
Project description:Amber suppression has been widely used to incorporate unnatural amino acids (UNAAs) with unique structures or functional side-chain groups into specific sites of the target protein, which expands the scope of protein-coding chemistry. However, this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein, which limits the development of unnatural proteins. To address this challenge, the suppression method using multiple termination codons (TAG, TAA or TGA) was proposed, and cell-free unnatural protein synthesis (CFUPS) system was employed. By the analysis of incorporating 3 different UNAAs (p-propargyloxy-l-phenylalanine, p-azyl-phenylalanine and L-4-Iodophenylalanine) and mass spectrometry, the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation. The CFUPS conditions were further optimized for better UNAA incorporation efficiency, including the orthogonal translation system (OTS) components, magnesium ions, and the redox environment. This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs, thereby synthesizing unnatural proteins with novel physicochemical functions.
| S-EPMC8718814 | biostudies-literature