Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.

Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 µmol/min/mg) of the activity of WT enzyme (95 µmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery.

Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema.

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.

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso.

BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcγRIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.

Structural origins for the loss of catalytic activities of bifunctional human LTA4H revealed through molecular dynamics simulations.

Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents.

Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections.

Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.

Leukotriene A4 metabolites are endogenous ligands for the Ah receptor.

In addition to orchestrating an adaptive metabolic response to xenobiotic compounds, the aryl hydrocarbon receptor (AHR) also plays a necessary role in the normal physiology of mice. The AHR is activated by a structurally diverse group of chemicals ranging from carcinogenic environmental pollutants to dietary metabolites and a number of endogenous molecules. Leukotriene A 4 (5,6-LTA 4) metabolites were identified in DRE-driven luciferase reporter assays as activators of AHR signaling. Various LTA 4 metabolites, including several 5,6- and 5,12-DiHETE products, were screened for AHR activity with 6- trans-LTB 4, 6- trans-12- epi-LTB 4, 5( S),6( S)-DiHETE, and 5( S),6( R)-DiHETE eliciting a significant level of AHR transcriptional activity. However, electrophoretic mobility shift assays (EMSAs) revealed that only 5,6-DiHETE isomers were capable of directly binding and activating the AHR to a DNA-binding species in vitro. Furthermore, ligand competition binding experiments confirm the ability of these compounds to directly bind to the AHR. Interestingly, "aged" preparations of 5,6-DiHETE isomers produced an enhanced level of AHR activation while demonstrating an increase in binding affinity for the receptor. Although the reason for this has not been fully determined, the formation of geometric isomers in the conjugated triene region of these molecules may play a role in the observed increase in AHR-mediated transcriptional activity. This work suggests a connection between AHR activation and inflammatory signaling molecules produced by the 5-lipoxygenase pathway.

Genetic susceptibility to myocardial infarction and coronary artery disease.

Atherosclerotic involvement in the coronary arteries, which can result in heart attack and sudden death, is a common disease and prototypic of a complex human trait. To understand its genomic basis, eight linkage studies of sibling pairs have been performed. Although there was limited inter-study concordance of important loci, two gene variants in the leukotriene pathway (ALOX5AP and LTA4) have emerged as susceptibility factors for myocardial infarction (MI). Genome-wide association studies have also been undertaken, and the pro-inflammatory cytokine lymphotoxin-alpha (LTA), and its key ligand galectin-2 (LGALS2) have been identified as genes implicated in predisposition for heart attack. By cueing into the genomic basis for low serum LDL cholesterol levels, much work has been done to advance the importance of the serine protease PCSK9, which modulates LDL receptor function. Lifelong lowered LDL cholesterol associated with PCSK9 point mutations in 2-3% of individuals have been shown to provide marked protection from coronary artery disease (CAD). Most of the success in this field has been with the phenotype of MI, which is considerably more restrictive than CAD. Four principal and interdependent processes--lipoprotein handling, endothelial integrity, arterial inflammation, and thrombosis--have been supported as important via the clustering of genes, thus far implicated in CAD susceptibility. Of note, connecting genes in a single pathway (leukotriene), of a protein and its ligand (LTAalpha) or from one disease to another [age-related macular degeneration (AMD); complement factor H (CFH)], or even three disease characterized by inflammation (MHC2) have now been reported. Although the population attributable risk for any of the genes identified to date is limited, such discovery is likely to be accelerated in the future.

Polymorphisms in inflammatory cytokines and Fcgamma receptors in childhood chronic immune thrombocytopenic purpura: a pilot study.

Inflammatory cytokines and low-affinity Fcgamma receptor (FcgammaR) polymorphisms were investigated in 37 children with chronic immune thrombocytopenic purpura (cITP) and 218 controls. Genotype analysis included common variants in the regulatory regions of cytokines, TNF, LTA, IL1RN, IL1A, IL1B, IL4, IL6 and IL10, and structural variants of the low affinity FcgammaRs, FCGR2A, FCGR3A and FCGR3B. Associations were observed for TNF (P = 0.0032), LTA (P = 0.019), FCGR3A (P = 0.038) and FCGR3B (P = 0.0034). Two combinations of genotypes (TNF and FCGR3A; P = 0.0003, and LTA and FCGR3B; P = 0.011) were significantly associated with cITP. These results provide preliminary evidence that variant genotypes of FcgammaRs and cytokines contribute to cITP pathogenesis.

Tumor necrosis factor is required for the priming of peritoneal macrophages by trehalose dimycolate.

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. We have developed an original model of macrophage activation where TDM is injected in vivo to prime peritoneal macrophages. These primed macrophages do not express inducible NO synthase (NOS II), however, they can be fully activated, i.e. induced to express NOS II and to develop a NOS II-dependent antiproliferative activity, following in vitro exposure to low concentrations of LPS. In a previous paper, we have shown that TDM-priming of mouse peritoneal macrophages is mediated by the sequential production of IL-12 and IFN-gamma. In the present paper, we investigated the role of TNF in the priming of macrophages by TDM. By semi-quantitative RT-PCR, we have shown that TDM injection induced transcription of TNF-alpha in peritoneal cells. TNF-mRNA levels peaked 5 hours after TDM injection and remained elevated for at least 32 hours. TNF expression was absolutely necessary for macrophage priming, as injection of an anti-TNF monoclonal antibody, 4 h before and 20 hours after TDM injection, prevented LPS-dependent activation of macrophages in vitro. This result was confirmed by the inability of TDM to prime macrophages from LT-alpha/TNF-alpha knockout (LT/TNFKO) mice. In addition, analysis of LT/TNFKO mice treated with TDM revealed that induction of the IL-12 transcript in their peritoneal cells and expression of a functional NADPH oxidase in macrophages are TNF-independent events.

Isolation of human promoter regions by Alu repeat consensus-based polymerase chain reaction.

Knowledge of the promoter structure is critical for an understanding of the regulation of genes. We demonstrate by analysis of 405 human genes that human promoter regions are flanked by upstream Alu repeat elements, typically at a distance of 0.5-5 kb from their protein-coding areas. We identified common Alu repeat consensus sequences (ARC) among the different members of the Alu subfamilies that can be used as universal anchor sites for polymerase chain reaction (PCR) amplification. Utilizing ARC-specific primers and oligonucleotides specific for the 5' end of a selected target gene, we show that sequences spanning unknown human gene promoter regions can be directly amplified by PCR from genomic DNA. This novel technique, termed ARC-PCR, allowed us to characterize the proximal promoters of the human LTA4 hydrolase and SPARC genes, each within 1 day.