ABSTRACT
Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Cell Nucleus/ultrastructure , Cytosol/ultrastructure , Protein Isoforms/metabolism , Arachidonate 5-Lipoxygenase/chemistry , Arachidonate 5-Lipoxygenase/isolation & purification , Cell Nucleus/metabolism , Cytosol/metabolism , Gene Expression Regulation, Enzymologic , HEK293 Cells , Humans , Leukotrienes/biosynthesis , Neutrophils/metabolism , Neutrophils/ultrastructure , Phosphorylation , Protein Isoforms/chemistry , Protein Isoforms/isolation & purificationABSTRACT
Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418.
Subject(s)
Cysteine/drug effects , Lipoxygenase Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Arachidonate 5-Lipoxygenase/chemistry , Arachidonate 5-Lipoxygenase/isolation & purification , Cell Line, Tumor , Chromatography, High Pressure Liquid , Enzyme Stability , Escherichia coli/metabolism , HEK293 Cells , Humans , Iron/metabolism , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Models, Molecular , Recombinant Proteins/metabolism , TemperatureABSTRACT
Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.
Subject(s)
5-Lipoxygenase-Activating Proteins/metabolism , Arachidonate 5-Lipoxygenase/metabolism , Cell Nucleus/metabolism , Cytosol/metabolism , Leukocytes, Mononuclear/metabolism , Leukotrienes/metabolism , 5-Lipoxygenase-Activating Proteins/chemistry , 5-Lipoxygenase-Activating Proteins/genetics , Amino Acid Substitution , Arachidonate 5-Lipoxygenase/chemistry , Arachidonate 5-Lipoxygenase/genetics , Arachidonic Acid/metabolism , Binding Sites , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Cytosol/drug effects , Cytosol/ultrastructure , Diamide/pharmacology , Gene Expression Regulation , Glutathione/metabolism , HEK293 Cells , HeLa Cells , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/ultrastructure , Mutation , Oxidation-Reduction , Primary Cell Culture , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal TransductionABSTRACT
Resolution of acute inflammation is an active process coordinated by proresolving lipid mediators (SPMs) such as lipoxins (LXs) and resolvins (Rvs), which are formed by the concerted action of 2 lipoxygenases (LOs). Because the exact molecular mechanisms of SPM biosynthesis are not completely understood, we aimed to investigate LX and D-type Rv formation in human leukocytes and HEK293T cells overexpressing leukotriene (LT) pathway enzymes. Activity assays in precursor (15-hydroxyeicosatetraenoic acids, 17-HDoHE)-treated granulocytes [polymorphonuclear leukocytes (PMNLs)] showed a strict dependence of LXA4/RvD1 biosynthesis on cell integrity, and incubation with recombinant human 5-LO did not lead to LX or Rv formation. Pharmacologic inhibition of 5-LO activating protein (FLAP) by MK-886 inhibited LXA4/RvD1 biosynthesis in precursor-treated PMNLs (drug concentration causing 50% inhibition â¼ 0.3/0.2 µM), as did knockdown of the enzyme in MM6 cells, and precursor-treated HEK293T overexpressing 5-LO produced high amounts of LXA4 only in the presence of FLAP. In addition, inhibition of cytosolic phospholipase A2α (cPLA2α) interfered with LXA4/RvD1 formation from exogenous precursors in PMNLs. Furthermore, inhibition of the LT synthases LTA4 hydrolase and LTC4 synthase in PMNL/platelet coincubations augmented LXA4 levels. These findings show that several enzymes known to be involved in the biosynthesis of proinflammatory LTs, such as FLAP and cPLA2α, also contribute to LX and Rv formation.