Dimerization of human 5-lipoxygenase

Häfner, Ann-Kathrin; Cernescu, Mihaela; Hofmann, Bettina; Ermisch, Michael; Hörnig, Michael; Metzner, Julia; Schneider, Gisbert; Brutschy, Bernhard; Steinhilber, Dieter

doi:10.1515/bc.2011.200

Cited by 49 publications

(41 citation statements)

References 64 publications

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“…Wild-type and C418S mutant (replacement of Cys418 to serine) 5-LO were purified by anion exchange chromatography as previously described (3,14) and used for the quantification of the iron content. Equal amounts (30 ng/reaction) of enzymes were incubated with solvent (1& DMSO), 5 lM of OA, LA, NO 2 -OA, or NO 2 -LA for 15 min, dialyzed overnight (Slide-A-Lyzer G2 dialysis cassette; Thermo Scientific) before HNO 3 (0.015 M final concentration) was added to each sample.…”

Section: Iron Determinationmentioning

confidence: 99%

“…The histidine residues modified in the 5-LO are conserved in the human platelet-type 12-LO and 15-LO-1 enzymes that are resistant to NO 2 -FA inhibition, inferring that the adduction of histidine residues alone could not account for preferential inhibition of 5-LO by NO 2 -FA. Cysteines at positions 159, 300, 416, and 418 are, on the other hand, unique to 5-LO; they are also located at the solvent interface of the enzyme and critical for enzymatic activity (14). The 5-LO tryptic peptide containing Cys416 and Cys418 was alkylated after incubation with NO 2 -OA (Table 2), and mass spectrometric analysis revealed that both cysteine residues were modified ( Fig.…”

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confidence: 99%

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Electrophilic Fatty Acid Species Inhibit 5-Lipoxygenase and Attenuate Sepsis-Induced Pulmonary Inflammation

Awwad

Steinbrink²,

Frömel³

et al. 2014

Antioxidants & Redox Signaling

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Aims: The reaction of nitric oxide and nitrite-derived species with polyunsaturated fatty acids yields electrophilic fatty acid nitroalkene derivatives (NO 2 -FA), which display anti-inflammatory properties. Given that the 5-lipoxygenase (5-LO, ALOX5) possesses critical nucleophilic amino acids, which are potentially sensitive to electrophilic modifications, we determined the consequences of NO 2 -FA on 5-LO activity in vitro and on 5-LOmediated inflammation in vivo. Results: Stimulation of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO 2 -OA) or nitro-linoleic acid (NO 2 -LA) (but not the parent lipids) resulted in the concentrationdependent and irreversible inhibition of 5-LO activity. Similar effects were observed in cell lysates and using the recombinant human protein, indicating a direct reaction with 5-LO. NO 2 -FAs did not affect the activity of the platelet-type 12-LO (ALOX12) or 15-LO-1 (ALOX15) in intact cells or the recombinant protein. The NO 2 -FAinduced inhibition of 5-LO was attributed to the alkylation of Cys418, and the exchange of Cys418 to serine rendered 5-LO insensitive to NO 2 -FA. In vivo, the systemic administration of NO 2 -OA to mice decreased neutrophil and monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5-hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. The administration of NO 2 -OA to 5-LO knockout mice had no effect on LPS-induced neutrophil or monocyte mobilization as well as on lung injury. Innovation: Prophylactic administration of NO 2 -OA to septic mice inhibits inflammation and promotes its resolution by interfering in 5-LO-mediated inflammatory processes. Conclusion: NO 2 -FAs directly and irreversibly inhibit 5-LO and attenuate downstream acute inflammatory responses. Antioxid. Redox Signal. 20, 2667Signal. 20, -2680

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Section: Iron Determinationmentioning

confidence: 99%

mentioning

confidence: 99%

Electrophilic Fatty Acid Species Inhibit 5-Lipoxygenase and Attenuate Sepsis-Induced Pulmonary Inflammation

Awwad

Steinbrink²,

Frömel³

et al. 2014

Antioxidants & Redox Signaling

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“…In silico phosphorylation of human 5-LO For visual inspection of the newly identified phosphorylation sites, a model of the human 5-LO crystal structure (Protein Data Bank, http://pdb.org [26]), Database ID 3o8y [40], was used as described previously [41]. Visualization was performed using PYMOL version 0.99rc6 (http://www.py mol.org/).…”

Section: Maldi-ms Analysismentioning

confidence: 99%

Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI‐MS

et al. 2014

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The enzyme 5-lipoxygenase (5-LO) catalyzes the first reactions in the biosynthesis of leukotrienes, powerful lipid mediators that are involved in several physiological and pathological processes. 5-LO activity is tightly regulated by several factors, including post translational modifications (PTMs). Phosphorylations of 5-LO by the kinases extracellular signal-regulated kinase 2 (Erk2), mitogen-activated protein kinase activated protein kinase 2 (MK2) and protein kinase A (PKA) have been described to regulate 5-LO activity. Furthermore, 5-LO phosphorylation is considered a determinant of drug candidate potency. However, no evidence on a molecular level, as can be provided by MS, has as yet been presented for these PTMs. Here, we employ a workflow including different proteolytic cleavages and phosphopeptide enrichment for detection of 5-LO phosphorylation by MALDI-MS. Proof for the known phosphorylation sites of MK2 (Ser271) and PKA (Ser523) was provided by MS after in vitro phosphorylation, but not for the postulated Erk2 site (Ser663). Detection limits have been determined for all three sites. Moreover, we identified novel tyrosine kinase target sites within 5-LO using in silico and in vitro methods. Tyr42, Tyr53 and either Tyr94 or Tyr445 were phosphorylated by the Src kinases Fgr, hematopoietic cell kinase (HCK) and Yes. To analyze the phosphorylation state in the cellular context, we created stably 5-LO-transduced Mono Mac 6 cells. Here, we only detected phospho-Ser271 by MS, whereas immunoblot analysis indicated tyrosine phosphorylation, phospho-Ser271 and phospho-Ser663. Unexpectedly, phospho-Ser271 occurred independent of cell stimulation. Taken together, we describe a method for the molecular analysis of 5-LO phosphorylation, provide insights regarding the occurrence of known phosphorylation sites partly in contrast to earlier studies and present first evidence on novel phosphosites.

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“…25 Lastly, the ability of 5-LOX to dimerize may provide yet another avenue of enzymatic regulation. 26 …”

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confidence: 99%

ATP Allosterically Activates the Human 5-Lipoxygenase Molecular Mechanism of Arachidonic Acid and 5(S)-Hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic Acid

et al. 2014

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5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

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Dimerization of human 5-lipoxygenase

Cited by 49 publications

References 64 publications

Electrophilic Fatty Acid Species Inhibit 5-Lipoxygenase and Attenuate Sepsis-Induced Pulmonary Inflammation

Electrophilic Fatty Acid Species Inhibit 5-Lipoxygenase and Attenuate Sepsis-Induced Pulmonary Inflammation

Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI‐MS

ATP Allosterically Activates the Human 5-Lipoxygenase Molecular Mechanism of Arachidonic Acid and 5(S)-Hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic Acid

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