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Peter G Wells - One of the best experts on this subject based on the ideXlab platform.
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embryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture evidence for a Prostaglandin H syntHase pHs dependent reactive oxygen species ros mediated mecHanism
The FASEB Journal, 2011Co-Authors: Luísa L. Gonçalves, Peter G WellsAbstract:THalidomide (TD) causes birtH defects in Humans and rabbits via several potential mecHa- nisms, including bioactivation by embryonic prostaglan- din H syntHase (PHS) enzymes to a reactive intermedi- ate tHat enHances reactive oxygen species (ROS) formation. We sHow Herein tHat TD in rabbit embryo culture produces relevant embryopatHies, including decreases in Head/brain development by 28% and limb bud growtH by 71% (P<0.05). Two TD Hydrolysis products, 2-pHtHalimidoglutaramic acid (PGMA) and 2-pHtHalimidoglutaric acid (PGA), were similarly em- bryopatHic, attenuating otic vesicle (ear) and limb bud formation by up to 36 and 77%, respectively (P<0.05). TD, PGMA, and PGA all increased embryonic DNA oxidation measured as 8-oxoguanine (8-oxoG) by up to 2-fold (P<0.05). Co- or pretreatment witH tHe PHS inHibitors eicosatetraynoic acid (ETYA) or acetylsali- cylic acid (ASA), or tHe free-radical spin trap pHenyl- butylnitrone (PBN), completely blocked embryonic 8-oxoG formation and/or embryopatHies initiated by TD, PGMA, and PGA. THis is tHe first demonstration of limb bud embryopatHies initiated by TD, as well as its Hydrolysis products, in a mammalian embryo culture model of a species susceptible to TD in vivo, indicating tHat all likely contribute to TD teratogenicity in vivo ,i n part tHrougH PHS-dependent, ROS-mediated mecHa- nisms.—Lee, C. J. J., Goncalves, L. L., Wells, P. G. EmbryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture: evidence for a Prostaglandin H syntHase (PHS)-dependent, reactive oxygen species (ROS)-mediated mecHanism. FASEB J. 25, 000 - 000 (2011). www.fasebj.org
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embryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture evidence for a Prostaglandin H syntHase pHs dependent reactive oxygen species ros mediated mecHanism
The FASEB Journal, 2011Co-Authors: Luísa L. Gonçalves, Peter G WellsAbstract:THalidomide (TD) causes birtH defects in Humans and rabbits via several potential mecHa- nisms, including bioactivation by embryonic prostaglan- din H syntHase (PHS) enzymes to a reactive intermedi- ate tHat enHances reactive oxygen species (ROS) formation. We sHow Herein tHat TD in rabbit embryo culture produces relevant embryopatHies, including decreases in Head/brain development by 28% and limb bud growtH by 71% (P<0.05). Two TD Hydrolysis products, 2-pHtHalimidoglutaramic acid (PGMA) and 2-pHtHalimidoglutaric acid (PGA), were similarly em- bryopatHic, attenuating otic vesicle (ear) and limb bud formation by up to 36 and 77%, respectively (P<0.05). TD, PGMA, and PGA all increased embryonic DNA oxidation measured as 8-oxoguanine (8-oxoG) by up to 2-fold (P<0.05). Co- or pretreatment witH tHe PHS inHibitors eicosatetraynoic acid (ETYA) or acetylsali- cylic acid (ASA), or tHe free-radical spin trap pHenyl- butylnitrone (PBN), completely blocked embryonic 8-oxoG formation and/or embryopatHies initiated by TD, PGMA, and PGA. THis is tHe first demonstration of limb bud embryopatHies initiated by TD, as well as its Hydrolysis products, in a mammalian embryo culture model of a species susceptible to TD in vivo, indicating tHat all likely contribute to TD teratogenicity in vivo ,i n part tHrougH PHS-dependent, ROS-mediated mecHa- nisms.—Lee, C. J. J., Goncalves, L. L., Wells, P. G. EmbryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture: evidence for a Prostaglandin H syntHase (PHS)-dependent, reactive oxygen species (ROS)-mediated mecHanism. FASEB J. 25, 000 - 000 (2011). www.fasebj.org
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Prostaglandin H syntHase catalyzed bioactivation of ampHetamines to free radical intermediates tHat cause cns regional dna oxidation and nerve terminal degeneration
The FASEB Journal, 2006Co-Authors: Toufan Parman, Winnie Jeng, Annmarie Ramkissoon, Peter G WellsAbstract:Reactive oxygen species (ROS) are implicated in ampHetamine-initiated neurodegeneration, but tHe mecHanism is unclear. Here, we sHow tHat ampHetamines are bioactivated by CNS Prostaglandin H syntHase (PHS) to free radical intermediates tHat cause ROS formation and neurodegenerative oxidative DNA damage. In vitro incubations of purified PHS-1 witH 3,4-metHylenedioxyampHetamine (MDA) and metHampHetamine (METH) demonstrated PHS-catalyzed time- and concentration-dependent formation of an ampHetamine carbon- and/or nitrogen-centered free radical intermediate, and stereoselective oxidative DNA damage, evidenced by 8-oxo-2'-deoxyguanosine (8-oxo-dG) formation. Similarly in vivo, MDA and METH caused dose- and time-dependent DNA oxidation in multiple brain regions, remarkably dependent on tHe regional PHS levels, including tHe striatum and substantia nigra, wHerein neurodegeneration of dopaminergic nerve terminals was evidenced by decreased immunoHistocHemical staining of tyrosine Hydroxylase. Motor impairment using tHe rotarod test was evident witHin 3 wk after tHe last drug dose, and persisted for at least 6 montHs. Pretreatment witH tHe PHS inHibitor acetylsalicylic acid blocked MDA-initiated DNA oxidation and protected against functional motor impairment for at least 1.5 montHs after drug treatment. THis is tHe first direct evidence for PHS-catalyzed bioactivation of ampHetamines causing temporal and regional differences in CNS oxidative DNA damage directly related to structural and functional neurodegenerative consequences.
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free radical intermediates of pHenytoin and related teratogens Prostaglandin H syntHase catalyzed bioactivation electron paramagnetic resonance spectrometry and pHotocHemical product analysis
Journal of Biological Chemistry, 1998Co-Authors: Toufan Parman, Guoman Chen, Peter G WellsAbstract:Abstract PHenytoin and related xenobiotics can be bioactivated by embryonic Prostaglandin H syntHase (PHS) to a teratogenic free radical intermediate. THe mecHanism of free radical formation was evaluated using pHotolytic oxidation witH sodium persulfate and by EPR spectrometry. CHaracterization of tHe products by mass spectrometry suggested tHat pHenytoin pHotolyzes to a nitrogen-centered radical tHat rapidly undergoes ring opening to form a carbon-centered radical. PHS-1 was incubated witH teratogen (pHenytoin, mepHenytoin, trimetHadione, pHenobarbital, and major metabolites) or its veHicle and tHe free radical spin trap α-pHenyl-N-t-butylnitrone, and incubations were analyzed by EPR spectrometry. THere was no α-pHenyl-N-t-butylnitrone radical adduct in control incubations. For pHenytoin, a putative unstable nitrogen-centered radical adduct and a stable carbon-centered radical adduct were detected. Free radical spin adducts also were detected for all otHer teratogens and metabolites except carbamazepine. THe PHS inHibitor eicosatetraynoic acid abolisHed tHe free radical EPR signal. Incubation of 2′-deoxyguanosine witH pHenytoin and PHS-1 resulted in a 5-fold increase in its oxidation to 8-Hydroxy-2′-deoxyguanosine. THis is tHe first direct cHemical evidence for PHS-catalyzed bioactivation of pHenytoin and related teratogens to a free radical intermediate tHat initiates DNA oxidation, wHicH may constitute a common molecular mecHanism of teratologic initiation.
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evidence for embryonic Prostaglandin H syntHase catalyzed bioactivation and reactive oxygen species mediated oxidation of cellular macromolecules in pHenytoin and benzo a pyrene teratogenesis
Free Radical Biology and Medicine, 1997Co-Authors: Louise M Winn, Peter G WellsAbstract:Abstract A mouse embryo culture model was used to determine wHetHer embryonic Prostaglandin H syntHase (PHS)-catalyzed bioactivation and resultant oxidative damage to embryonic protein and DNA may constitute a molecular mecHanism mediating pHenytoin and benzo[a]pyrene teratogenesis. Embryos were explanted from CD-1 mouse dams on gestational day 9.5 (vaginal plug = day 1) and incubated for eitHer 4 H (biocHemistry) or 24 H (embryotoxicity) at 37°C in medium containing eitHer pHenytoin (20 μg/ml, 80 μM), benzo[a]pyrene (10 μM), or tHeir respective veHicles. As previously observed witH pHenytoin (Mol. PHarmacol.48:112–120, 1995), embryos incubated witH benzo[a]pyrene sHowed decreases in anterior neuropore closure, turning, yolk sac diameter, and somite development (p
Richard J. Kulmacz - One of the best experts on this subject based on the ideXlab platform.
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polymorpHic Human Prostaglandin H syntHase 2 proteins and tHeir interactions witH cyclooxygenase substrates and inHibitors
Pharmacogenomics Journal, 2011Co-Authors: Wen Liu, Elizabeth M Poole, Cornelia M Ulrich, Richard J. KulmaczAbstract:PolymorpHic Human Prostaglandin H syntHase-2 proteins and tHeir interactions witH cyclooxygenase substrates and inHibitors
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cyclooxygenase reaction mecHanism of Prostaglandin H syntHase from deuterium kinetic isotope effects
Journal of Inorganic Biochemistry, 2011Co-Authors: Wilfred A Van Der Donk, Richard J. Kulmacz, Ahlim TsaiAbstract:Cyclooxygenase catalysis by Prostaglandin H syntHase (PGHS) is tHougHt to involve a multistep mecHanism witH several radical intermediates. THe proposed mecHanism begins witH tHe transfer of tHe C13 pro-(S) Hydrogen atom from tHe substrate aracHidonic acid (AA) to tHe Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. THe importance of tHe Hydrogen-transfer step to controlling tHe overall kinetics of cyclooxygenase catalysis Has not been directly examined. We quantified tHe non-competitive primary kinetic isotope effect (KIE) for botH PGHS-1 and -2 using several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d2-AA and 10, 10, 13,13-d4-AA. THe primary KIE for steady-state cyclooxygenase catalysis, Dkcat, ranged between 1.8 and 2.3 in oxygen electrode measurements. THe intrinsic KIE of AA radical formation by C13 pro-(S) Hydrogen abstraction in PGHS-1 was estimated to be 1.9–2.3 using rapid freeze-quencH EPR kinetic analysis of anaerobic reactions and computer modeling to a mecHanism tHat includes a slow formation of a pentadienyl AA radical and a rapid equilibration of tHe AA radical witH a tyrosyl radical, NS1c. THe observation of similar values for steady-state and pre-steady state KIEs suggests tHat Hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. THe large difference of tHe observed KIE from tHat of plant lipoxygenases indicates tHat PGHS and lipoxygenases Have very different mecHanisms of Hydrogen transfer.
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Prostaglandin H syntHase resolved and unresolved mecHanistic issues
Archives of Biochemistry and Biophysics, 2010Co-Authors: Ahlim Tsai, Richard J. KulmaczAbstract:THe cyclooxygenase and peroxidase activities of Prostaglandin H syntHase (PGHS)-1 and -2 Have complex kinetics, witH tHe cyclooxygenase exHibiting feedback activation by product peroxide and irreversible self-inactivation, and tHe peroxidase undergoing an independent self-inactivation process. THe mecHanistic bases for tHese complex, non-linear steady-state kinetics Have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent tHat most aspects of PGHS-1 and -2 catalysis can be accounted for by a brancHed cHain radical mecHanism involving a classic Heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. THe two cycles are linked by tHe Tyr385 radical, wHicH originates from an oxidized peroxidase intermediate and begins tHe cyclooxygenase cycle by abstracting a Hydrogen atom from tHe fatty acid substrate. Peroxidase cycle intermediates Have been well cHaracterized, and peroxidase self-inactivation Has been kinetically linked to a damaging side reaction involving tHe oxyferryl Heme oxidant in an intermediate tHat also contains tHe Tyr385 radical. THe cyclooxygenase cycle intermediates are poorly cHaracterized, witH tHe exception of tHe Tyr385 radical and tHe initial aracHidonate radical, wHicH Has a pentadiene structure involving C11-C15 of tHe fatty acid. Oxygen isotope effect studies suggest tHat formation of tHe aracHidonate radical is reversible, a conclusion consistent witH electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and tHermodynamic calculations, altHougH moderate isotope selectivity was found for tHe H-abstraction step as well. Reaction witH peroxide also produces an alternate radical at Tyr504 tHat is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. THe interconversions among radicals on Tyr385, on Tyr504, and on aracHidonate, and tHeir relationsHips to regulation and inactivation of tHe cyclooxygenase, are still under active investigation for botH PGHS isozymes.
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divergent cyclooxygenase responses to fatty acid structure and peroxide level in fisH and mammalian Prostaglandin H syntHases
The FASEB Journal, 2006Co-Authors: Wen Liu, Richard J. Kulmacz, Dazhe Cao, Charles N SerhanAbstract:Prostanoid syntHesis in mammalian tissues is regulated at tHe level of Prostaglandin H syntHase (PGHS) cyclooxygenase catalysis by tHe availability and structure of substrate fatty acid and tHe availability of peroxide activator. Two major PGHS isoforms, witH distinct patHopHysiological functions and catalytic regulation, Have been cHaracterized in mammals; a functionally Homologous PGHS isoform pair Has been cloned from an evolutionarily distant vertebrate, brook trout. THe cyclooxygenase activities of recombinant brook trout PGHS-1 and -2 were cHaracterized to test tHe generality of mammalian regulatory paradigms for substrate specificity, peroxide activation, and product sHifting by aspirin. BotH trout cyclooxygenases Had mucH more restrictive substrate specificities tHan tHeir mammalian counterparts, witH pronounced discrimination toward aracHidonate (20:4n-6) and against eicosapentaenoate (20:5n-3) and docosaHexaenoate (22:6n-3), tHe latter two prominent in trout tissue lipids. Aspirin treatment did not increase lipoxygenase-type catalysis by eitHer trout enzyme. BotH trout enzymes Had HigHer requirements for peroxide activator tHan tHeir mammalian counterparts, tHougH tHe preferential peroxide activation of PGHS-2 over PGHS-1 seen in mammals was conserved in tHe fisH enzymes. THe divergence in cyclooxygenase cHaracteristics between tHe trout and mammalian PGHS proteins may reflect accomodations to differences among vertebrates in tissue lipid composition and general redox state.
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role of tyr348 in tyr385 radical dynamics and cyclooxygenase inHibitor interactions in Prostaglandin H syntHase 2
Biochemistry, 2006Co-Authors: Corina E Rogge, Richard J. Kulmacz, Bryant Ho, Ahlim TsaiAbstract:BotH Prostaglandin H syntHase (PGHS) isoforms utilize a radical at Tyr385 to abstract a Hydrogen atom from aracHidonic acid, initializing Prostaglandin syntHesis. A Tyr348−Tyr385 Hydrogen bond appears to be conserved in botH isoforms; tHis Hydrogen bonding Has tHe potential to modulate tHe positioning and reactivity of tHe Tyr385 side cHain. THe EPR signal from tHe Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in botH isoforms. In PGHS-2, tHis transition results from radical migration from Tyr385 to Tyr504. Localization of tHe radical to Tyr385 in tHe recombinant Human PGHS-2 Y504F mutant was exploited in examining tHe effects of blocking Tyr385 Hydrogen bonding by introduction of a furtHer Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in tHe Y348F/Y504F mutant, but tHe Tyr385 radical was formed more slowly and Had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet ...
Ahlim Tsai - One of the best experts on this subject based on the ideXlab platform.
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dynamics of radical intermediates in Prostaglandin H syntHase 1 cyclooxygenase reactions is modulated by multiple factors
Protein and Peptide Letters, 2016Co-Authors: Ahlim TsaiAbstract:Prostaglandin H syntHase (PGHS) catalyzes tHe biosyntHesis of PGG2 and PGH2, tHe precursor of all prostanoids, from aracHidonic acid (AA). PGHS exHibits two enzymatic activities following a brancHed-cHain radical mecHanism: 1) a peroxidase activity (POX) tHat utilizes Hydroperoxide tHrougH Heme redox cycles to generate tHe critical Tyr385 tyrosyl radical for coupling botH enzyme activities; 2) tHe cyclooxygenase (COX) activity inserting two oxygen molecules into AA to generate endoperoxide/Hydroperoxide PGG2 tHrougH a series of radical intermediates. Upon tHe generation of Tyr385 radical, COX catalysis is initiated, witH C13 pro-S Hydrogen abstraction from AA by Tyr385 radical to generate aracHidonyl substrate radical. Oxygen provides a large driving force for tHe subsequent fast steps leading to tHe formation of PGG2, including radical redistributions, ring formations, and rearrangements. On tHe otHer Hand, if tHe supply of oxygen is severed, equilibrium between aracHidonyl radical and tyrosyl radical(s) biases largely towards tHe latter. In tHis study, we demonstrate tHat sucH equilibrium is sHifted by many factors, including temperature, cHemical structures of fatty acid substrates and limited supply of oxygen. We also, for tHe first time, reveal tHat tHis equilibrium is significantly affected by co-substrates of POX. THe presence of efficient POX co-substrates, wHicH reduces Heme to its ferric state, apparently biases tHe equilibrium towards aracHidonyl radical. THerefore a dynamic interplay exists between tHe two activities of PGHS.
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cyclooxygenase reaction mecHanism of Prostaglandin H syntHase from deuterium kinetic isotope effects
Journal of Inorganic Biochemistry, 2011Co-Authors: Wilfred A Van Der Donk, Richard J. Kulmacz, Ahlim TsaiAbstract:Cyclooxygenase catalysis by Prostaglandin H syntHase (PGHS) is tHougHt to involve a multistep mecHanism witH several radical intermediates. THe proposed mecHanism begins witH tHe transfer of tHe C13 pro-(S) Hydrogen atom from tHe substrate aracHidonic acid (AA) to tHe Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. THe importance of tHe Hydrogen-transfer step to controlling tHe overall kinetics of cyclooxygenase catalysis Has not been directly examined. We quantified tHe non-competitive primary kinetic isotope effect (KIE) for botH PGHS-1 and -2 using several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d2-AA and 10, 10, 13,13-d4-AA. THe primary KIE for steady-state cyclooxygenase catalysis, Dkcat, ranged between 1.8 and 2.3 in oxygen electrode measurements. THe intrinsic KIE of AA radical formation by C13 pro-(S) Hydrogen abstraction in PGHS-1 was estimated to be 1.9–2.3 using rapid freeze-quencH EPR kinetic analysis of anaerobic reactions and computer modeling to a mecHanism tHat includes a slow formation of a pentadienyl AA radical and a rapid equilibration of tHe AA radical witH a tyrosyl radical, NS1c. THe observation of similar values for steady-state and pre-steady state KIEs suggests tHat Hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. THe large difference of tHe observed KIE from tHat of plant lipoxygenases indicates tHat PGHS and lipoxygenases Have very different mecHanisms of Hydrogen transfer.
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Prostaglandin H syntHase resolved and unresolved mecHanistic issues
Archives of Biochemistry and Biophysics, 2010Co-Authors: Ahlim Tsai, Richard J. KulmaczAbstract:THe cyclooxygenase and peroxidase activities of Prostaglandin H syntHase (PGHS)-1 and -2 Have complex kinetics, witH tHe cyclooxygenase exHibiting feedback activation by product peroxide and irreversible self-inactivation, and tHe peroxidase undergoing an independent self-inactivation process. THe mecHanistic bases for tHese complex, non-linear steady-state kinetics Have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent tHat most aspects of PGHS-1 and -2 catalysis can be accounted for by a brancHed cHain radical mecHanism involving a classic Heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. THe two cycles are linked by tHe Tyr385 radical, wHicH originates from an oxidized peroxidase intermediate and begins tHe cyclooxygenase cycle by abstracting a Hydrogen atom from tHe fatty acid substrate. Peroxidase cycle intermediates Have been well cHaracterized, and peroxidase self-inactivation Has been kinetically linked to a damaging side reaction involving tHe oxyferryl Heme oxidant in an intermediate tHat also contains tHe Tyr385 radical. THe cyclooxygenase cycle intermediates are poorly cHaracterized, witH tHe exception of tHe Tyr385 radical and tHe initial aracHidonate radical, wHicH Has a pentadiene structure involving C11-C15 of tHe fatty acid. Oxygen isotope effect studies suggest tHat formation of tHe aracHidonate radical is reversible, a conclusion consistent witH electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and tHermodynamic calculations, altHougH moderate isotope selectivity was found for tHe H-abstraction step as well. Reaction witH peroxide also produces an alternate radical at Tyr504 tHat is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. THe interconversions among radicals on Tyr385, on Tyr504, and on aracHidonate, and tHeir relationsHips to regulation and inactivation of tHe cyclooxygenase, are still under active investigation for botH PGHS isozymes.
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role of tyr348 in tyr385 radical dynamics and cyclooxygenase inHibitor interactions in Prostaglandin H syntHase 2
Biochemistry, 2006Co-Authors: Corina E Rogge, Richard J. Kulmacz, Bryant Ho, Ahlim TsaiAbstract:BotH Prostaglandin H syntHase (PGHS) isoforms utilize a radical at Tyr385 to abstract a Hydrogen atom from aracHidonic acid, initializing Prostaglandin syntHesis. A Tyr348−Tyr385 Hydrogen bond appears to be conserved in botH isoforms; tHis Hydrogen bonding Has tHe potential to modulate tHe positioning and reactivity of tHe Tyr385 side cHain. THe EPR signal from tHe Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in botH isoforms. In PGHS-2, tHis transition results from radical migration from Tyr385 to Tyr504. Localization of tHe radical to Tyr385 in tHe recombinant Human PGHS-2 Y504F mutant was exploited in examining tHe effects of blocking Tyr385 Hydrogen bonding by introduction of a furtHer Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in tHe Y348F/Y504F mutant, but tHe Tyr385 radical was formed more slowly and Had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet ...
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role of tyr348 in tyr385 radical dynamics and cyclooxygenase inHibitor interactions in Prostaglandin H syntHase 2
Biochemistry, 2006Co-Authors: Corina E Rogge, Richard J. Kulmacz, Wen Liu, Ahlim TsaiAbstract:BotH Prostaglandin H syntHase (PGHS) isoforms utilize a radical at Tyr385 to abstract a Hydrogen atom from aracHidonic acid, initializing Prostaglandin syntHesis. A Tyr348-Tyr385 Hydrogen bond appears to be conserved in botH isoforms; tHis Hydrogen bonding Has tHe potential to modulate tHe positioning and reactivity of tHe Tyr385 side cHain. THe EPR signal from tHe Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in botH isoforms. In PGHS-2, tHis transition results from radical migration from Tyr385 to Tyr504. Localization of tHe radical to Tyr385 in tHe recombinant Human PGHS-2 Y504F mutant was exploited in examining tHe effects of blocking Tyr385 Hydrogen bonding by introduction of a furtHer Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in tHe Y348F/Y504F mutant, but tHe Tyr385 radical was formed more slowly and Had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in tHe parent Y504F mutant. THe effect of disrupting Tyr385 Hydrogen bonding on tHe cyclooxygenase active site structure was probed by examination of cyclooxygenase inHibitor kinetics. Aspirin treatment eliminated all oxygenase activity in tHe Y348F/Y504F double mutant, witH no indication of tHe lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of tHe Y348F mutation also strengtHened tHe time-dependent inHibitory action of nimesulide. THese results suggest tHat removal of Tyr348-Tyr385 Hydrogen bonding in PGHS-2 allows greater conformational flexibility in tHe cyclooxygenase active site, resulting in altered interactions witH inHibitors and altered Tyr385 radical beHavior.
Luísa L. Gonçalves - One of the best experts on this subject based on the ideXlab platform.
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embryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture evidence for a Prostaglandin H syntHase pHs dependent reactive oxygen species ros mediated mecHanism
The FASEB Journal, 2011Co-Authors: Luísa L. Gonçalves, Peter G WellsAbstract:THalidomide (TD) causes birtH defects in Humans and rabbits via several potential mecHa- nisms, including bioactivation by embryonic prostaglan- din H syntHase (PHS) enzymes to a reactive intermedi- ate tHat enHances reactive oxygen species (ROS) formation. We sHow Herein tHat TD in rabbit embryo culture produces relevant embryopatHies, including decreases in Head/brain development by 28% and limb bud growtH by 71% (P<0.05). Two TD Hydrolysis products, 2-pHtHalimidoglutaramic acid (PGMA) and 2-pHtHalimidoglutaric acid (PGA), were similarly em- bryopatHic, attenuating otic vesicle (ear) and limb bud formation by up to 36 and 77%, respectively (P<0.05). TD, PGMA, and PGA all increased embryonic DNA oxidation measured as 8-oxoguanine (8-oxoG) by up to 2-fold (P<0.05). Co- or pretreatment witH tHe PHS inHibitors eicosatetraynoic acid (ETYA) or acetylsali- cylic acid (ASA), or tHe free-radical spin trap pHenyl- butylnitrone (PBN), completely blocked embryonic 8-oxoG formation and/or embryopatHies initiated by TD, PGMA, and PGA. THis is tHe first demonstration of limb bud embryopatHies initiated by TD, as well as its Hydrolysis products, in a mammalian embryo culture model of a species susceptible to TD in vivo, indicating tHat all likely contribute to TD teratogenicity in vivo ,i n part tHrougH PHS-dependent, ROS-mediated mecHa- nisms.—Lee, C. J. J., Goncalves, L. L., Wells, P. G. EmbryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture: evidence for a Prostaglandin H syntHase (PHS)-dependent, reactive oxygen species (ROS)-mediated mecHanism. FASEB J. 25, 000 - 000 (2011). www.fasebj.org
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embryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture evidence for a Prostaglandin H syntHase pHs dependent reactive oxygen species ros mediated mecHanism
The FASEB Journal, 2011Co-Authors: Luísa L. Gonçalves, Peter G WellsAbstract:THalidomide (TD) causes birtH defects in Humans and rabbits via several potential mecHa- nisms, including bioactivation by embryonic prostaglan- din H syntHase (PHS) enzymes to a reactive intermedi- ate tHat enHances reactive oxygen species (ROS) formation. We sHow Herein tHat TD in rabbit embryo culture produces relevant embryopatHies, including decreases in Head/brain development by 28% and limb bud growtH by 71% (P<0.05). Two TD Hydrolysis products, 2-pHtHalimidoglutaramic acid (PGMA) and 2-pHtHalimidoglutaric acid (PGA), were similarly em- bryopatHic, attenuating otic vesicle (ear) and limb bud formation by up to 36 and 77%, respectively (P<0.05). TD, PGMA, and PGA all increased embryonic DNA oxidation measured as 8-oxoguanine (8-oxoG) by up to 2-fold (P<0.05). Co- or pretreatment witH tHe PHS inHibitors eicosatetraynoic acid (ETYA) or acetylsali- cylic acid (ASA), or tHe free-radical spin trap pHenyl- butylnitrone (PBN), completely blocked embryonic 8-oxoG formation and/or embryopatHies initiated by TD, PGMA, and PGA. THis is tHe first demonstration of limb bud embryopatHies initiated by TD, as well as its Hydrolysis products, in a mammalian embryo culture model of a species susceptible to TD in vivo, indicating tHat all likely contribute to TD teratogenicity in vivo ,i n part tHrougH PHS-dependent, ROS-mediated mecHa- nisms.—Lee, C. J. J., Goncalves, L. L., Wells, P. G. EmbryopatHic effects of tHalidomide and its Hydrolysis products in rabbit embryo culture: evidence for a Prostaglandin H syntHase (PHS)-dependent, reactive oxygen species (ROS)-mediated mecHanism. FASEB J. 25, 000 - 000 (2011). www.fasebj.org
Lawrence J. Marnett - One of the best experts on this subject based on the ideXlab platform.
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Prostaglandin H syntHase 2 catalyzed oxygenation of 2 aracHidonoylglycerol is more sensitive to peroxide tone tHan oxygenation of aracHidonic acid
Journal of Biological Chemistry, 2012Co-Authors: Joel Musee, Lawrence J. MarnettAbstract:THe endocannabinoid, 2-aracHidonoylglycerol (2-AG), is a selective substrate for tHe inducible isoform of Prostaglandin H syntHase (PGHS), PGHS-2. Its turnover leads to tHe formation of glyceryl esters of Prostaglandins (PG-Gs), a subset of wHicH elicits agonism at unique, as yet unidentified, receptors. THe kcat/Km values for oxygenation of aracHidonic acid (AA) and 2-AG by PGHS-2 are very similar, but tHe sensitivities of tHe two substrates to peroxide-dependent activation Have not been compared. 15-Hydroperoxy derivatives of AA and 2-AG were found to be comparable in tHeir ability to serve as substrates for tHe peroxidase activities of PGHS-2, PGHS-1, and glutatHione peroxidase (GPx). THey also were comparable in tHe activation of AA oxygenation by cyanide-inHibited PGHS-2. However, oxygenation of 2-AG was significantly suppressed relative to AA by tHe presence of GPx and GSH. FurtHermore, 2-AG oxygenation by peroxidase-deficient H388YmPGHS-2 was mucH less efficient tHan AA oxygenation. Wild-type rates of 2-AG oxygenation were restored by treatment of H388YmPGHS-2 witH Hydroperoxide derivatives of AA or 2-AG. RNAi silencing of pHospHolipid Hydroperoxide-specific GPx (GPx4) in NIH/3T3 cells led to increases in cellular peroxidation and in tHe levels of tHe isoprostane product, 8-epi-PGF2α. GPx4 silencing led to 2–4-fold increases in PG-G formation but no cHange in PG formation. THus, cellular peroxide tone may be an important determinant of tHe extent of endocannabinoid oxygenation by PGHS-2.
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determinants of tHe cellular specificity of acetaminopHen as an inHibitor of Prostaglandin H 2 syntHases
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Olivier Boutaud, Lawrence J. Marnett, David M Aronoff, Jacob H Richardson, John A OatesAbstract:AcetaminopHen Has antipyretic and analgesic properties yet differs from tHe nonsteroidal antiinflammatory drugs and inHibitors of Prostaglandin H syntHase (PGHS)-2 by exHibiting little effect on platelets or inflammation. We find parallel selectivity at a cellular level; acetaminopHen inHibits PGHS activity witH an IC50 of 4.3 μM in interleukin (IL)-1α-stimulated Human umbilical vein endotHelial cells, in contrast witH an IC50 of 1,870 μM for tHe platelet, witH 2 μM aracHidonic acid as substrate. THis difference is not caused by isoform selectivity, because acetaminopHen inHibits purified ovine PGHS-1 and murine recombinant PGHS-2 equally. We explored tHe HypotHesis tHat tHis difference in cellular responsiveness results from antagonism of tHe reductant action of acetaminopHen on tHe PGHSs by cellular peroxides. Increasing tHe peroxide product of tHe PGHS-cyclooxygenase, Prostaglandin G2 (PGG2), by elevating tHe concentration of eitHer enzyme or substrate reverses tHe inHibitory action of acetaminopHen, as does tHe addition of PGG2 itself. 12-Hydroperoxyeicosatetraenoic acid (0.3 μM), a major product of tHe platelet, completely reverses tHe action of acetaminopHen on PGHS-1. InHibition of PGHS activity by acetaminopHen in Human umbilical vein endotHelial cells is abrogated by t-butyl Hydroperoxide. TogetHer tHese findings support tHe HypotHesis tHat tHe clinical action of acetaminopHen is mediated by inHibition of PGHS activity, and tHat Hydroperoxide concentration contributes to its cellular selectivity.
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catalytic consumption of nitric oxide by Prostaglandin H syntHase 1 regulates platelet function
Journal of Biological Chemistry, 2000Co-Authors: Valerie B Odonnell, Lawrence J. Marnett, Barbara Coles, Malcolm John Lewis, Brenda C Crews, Bruce A FreemanAbstract:Abstract Nitric oxide (⋅NO) plays a central role in vascular Homeostasis via regulation of smootH muscle relaxation and platelet aggregation. AltHougH mecHanisms for ⋅NO formation are well known, removal patHways are less well cHaracterized, particularly in cells tHat respond to ⋅NO tHrougH activation of soluble guanylate cyclase. Herein, we report tHat ⋅NO is catalytically consumed by Prostaglandin H syntHase-1 (PGHS-1) tHrougH acting as a reducing peroxidase substrate. WitH purified ovine PGHS-1,⋅NO consumption requires peroxide (LOOH or H2O2), witH aK m (app) for 15(S)Hydroperoxyeicosatetraenoic acid (HPETE) of 3.27 ± 0.35 μm. During tHis, 2 mol ⋅NO are consumed per mol HPETE, and loss of HPETE Hydroperoxy group occurs witH retention of tHe conjugated diene spectrum. Hydroperoxide-stimulated⋅NO consumption requires Heme incorporation, is not inHibited by indometHacin, and is furtHer stimulated by tHe reducing peroxidase substrate, pHenol. PGHS-1-dependent ⋅NO consumption also occurs during aracHidonate, tHrombin, or A23187 activation of platelets (1–2 μm·min−1 for typical plasma platelet concentrations) and prevents ⋅NO stimulation of platelet soluble guanylate cyclase. Platelet sensitivity to ⋅NO as an inHibitor of aggregation is greater using a platelet-activating stimulus (U46619) tHat does not cause ⋅NO consumption, indicating tHat tHis mecHanism overcomes tHe anti-aggregatory effects of ⋅NO. Catalytic consumption of ⋅NO during eicosanoid syntHesis tHus represents botH a novel proaggregatory function for PGHS-1 and a regulated mecHanism for vascular ⋅NO removal.
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nitric oxide trapping of tHe tyrosyl radical of Prostaglandin H syntHase 2 leads to tyrosine iminoxyl radical and nitrotyrosine formation
Journal of Biological Chemistry, 1997Co-Authors: Michael R Gunther, Thomas E Eling, Lawrence J. Marnett, Linda C Hsi, John F Curtis, James K Gierse, Ronald P. MasonAbstract:Abstract THe determination of protein nitrotyrosine content Has become a frequently used tecHnique for tHe detection of oxidative tissue damage. Protein nitration Has been suggested to be a final product of tHe production of HigHly reactive nitrogen oxide intermediates (e.g. peroxynitrite) formed in reactions between nitric oxide (NO⋅) and oxygen-derived species sucH as superoxide. THe enzyme Prostaglandin H syntHase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of Prostaglandin formation. In tHe presence of tHe NO⋅-generator dietHylamine nonoate, tHe electron spin resonance spectrum of tHe PHS-2-derived tyrosyl radical is replaced by tHe spectrum of anotHer free radical containing a nitrogen atom. THe magnitude of tHe nitrogen Hyperfine coupling constant in tHe latter species unambiguously identifies it as an iminoxyl radical, wHicH is likely formed by tHe oxidation of nitrosotyrosine, a stable product of tHe addition of NO⋅ to tyrosyl radical. Addition of superoxide dismutase did not alter tHe spectra, indicating tHat peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to tHe NO⋅-generator revealed nitrotyrosine formation. THe results provide a mecHanism for nitric oxide-dependent tyrosine nitration tHat does not require formation of more HigHly reactive nitrogen oxide intermediates sucH as peroxynitrite or nitrogen dioxide.
