Functional role of the conserved i-helix residue I346 in CYP5A1-Nanodiscs.

Thromboxane synthase (CYP5A1) is a non-classical cytochrome P450 (CYP) expressed in human platelets that mediates vascular homeostasis by producing thromboxane A2 (TXA2) through the isomerization of prostaglandin H2 (PGH2). A homology alignment of CYP5A1 with human CYPs indicates that a highly conserved I-helix threonine residue is occupied by an isoleucine at position 346 in CYP5A1. We find that reverse-engineering CYP5A1 to contain either threonine or serine in this position dramatically increases TXA2 formation. Interestingly, the levels of malondialdehyde (MDA), a homolytic fragmentation product of PGH2 formed via a pathway independent of TXA2 formation, remain constant. Furthermore, spectral analysis using two PGH2 substrate analogs supports the observed activity changes in the hydroxyl-containing mutants. The more constrained active site of the I346T mutant displays altered PGH2 substrate analog binding properties. Together these studies provide new mechanistic insights into CYP5A1 mediated isomerization of PGH2 with respect to a critical active site residue.

Functional investigations of thromboxane synthase (CYP5A1) in lipid bilayers of nanodiscs.

CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2 ) into thromboxane A2 (TXA2 ), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.

O endotélio disfuncionante na hipertensão arterial e no tabagismo / Dysfunctional endothelium in hypertension and smoking

Disfunção endotelial (DE) caracteriza qualquer alteração de atividadenormal do endotélio incluindo atividade vasomotora, proliferaçãocelular, adesão/agregação plaquetária, permeabilidade vascular e a interaçãoleucócitos/parede vascular. Contudo, em repouso o leito arterialexibe um estado basal de vasoconstrição (tônus vascular) modulado pormecanismos de controle centrais (sistema nervoso-simpático), periféricos(sistema renina-angiotensina-aldosterona) e um mecanismo local(endotelial) cuja potência é superior às anteriores. DE na hipertensãoestá relacionada à diminuição da biodisponibilidade de NO por reduçãoda síntese e liberação pela sintase endotelial do óxido nítrico (eNOS)influenciada por fatores genéticos e ambientais como hipóxia, hipofluxo,forças de cisalhamento, redução do substrato L-arginina e seuscofatores, ou inativação do NO resultado da sua ligação com diferentesmoléculas hemoglobina, albumina e, principalmente, sua interação comespécies reativas de oxigênio (estresse oxidativo). DE na hipertensãopode, ainda, estar associada à liberação de substâncias vasoconstritorasderivadas do endotélio como trornboxano-Aç, prostaglândina-Hj,endotelina-l e angotensina-Il. Tabagismo aumenta o risco de eventoscardiovasculares, principalmente quando associado à hipertensão.Nicotina estimula a liberação de catecolarninas e promove lesões noendotélio vascular. Radicais livres e compostos aromáticos diminuema síntese de NO, prejudicando a vasodilatação endotélio-dependente.Tabagismo favorece o crescimento celular por estimulação de fatoresde crescimento do endotélio vascular e inativação do NO por aumentodo estresse oxidativo. Aumento da oxidação das lipoproteínas debaixa densidade (LDL) em fumantes tem efeito sinérgico na adesão emigração de monócitos. Todos esses efeitos deletérios do tabagismono leito vascular também são observados, embora em menor extensão,em tabagistas passivos...

Endothelial dysfunction (ED) characterizes ali changes in the normalactivity ofthe endothelium including vasomotor activity, cell proliferation,platelet adhesion and aggregation, vascular permeability andleukocyte-vascular wall interactions. However, at rest, the arterialbed exhibits a baseline state of vasoconstriction (vascular tone) whichis modulated by central control (sympathetic nervous system) andperipheral mechanisms (Renin angiotensin-aldosterone system) anda local mechanism (endothelial); the latter is the most potent. ED inhypertension is related to decreased bioavailability of nitric oxide(NO) due to its reduced synthesis and release by endothelial nitricoxide synthase (eNOS) regarding genetic and environmental factorssuch as hypoxia, decreased blood flow, shear forces, diminishedL-arginine substrate and its cofactors and NO inactivation as a resultof its binding to different molecules including hemoglobin, albuminand, mainly, its interaction with reactive oxygen species (oxidativestress). ED in hypertension can also be linked to the release of endothelium-derived vasoconstricting substances such as thromboxane-Ai,prostaglandin-H2, endothelin-l and angiotensin-Il. Smoking increasesthe risk of cardiovascular events especially when associated withhypertension. Nicotine stimulates catecholamine release and causesvascular endothelium injury. Free radicais and aromatic compoundsreduce NO synthesis, impairing endothelium-dependent vasodilation.Smoking promotes cell growth by stimulating vascular endotheliumgrowth factors and NO inactivation by increasing oxidative stress.The increased oxidation of low-density lipoproteins ir smokers hasa synergistic effect on monocyte adhesion and migration. All thesedeleterious effects of smoking on the vascular bed are also observed,albeit at a lesser extent, in passive smokers...

Probing water micro-solvation in proteins by water catalysed proton-transfer tautomerism.

Scientists have made tremendous efforts to gain understanding of the water molecules in proteins via indirect measurements such as molecular dynamic simulation and/or probing the polarity of the local environment. Here we present a tryptophan analogue that exhibits remarkable water catalysed proton-transfer properties. The resulting multiple emissions provide unique fingerprints that can be exploited for direct sensing of a site-specific water environment in a protein without disrupting its native structure. Replacing tryptophan with the newly developed tryptophan analogue we sense different water environments surrounding the five tryptophans in human thromboxane A2 synthase. This development may lead to future research to probe how water molecules affect the folding, structures and activities of proteins.

Probing ligand binding to thromboxane synthase.

Various fluorescence experiments and computer simulations were utilized to gain further understanding of thromboxane A(2) synthase (TXAS), which catalyzes an isomerization of prostaglandins H(2) to give rise to thromboxane A(2) along with a fragmentation reaction to 12-L-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde. In this study, 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS) was utilized as a probe to assess the spatial relationship and binding dynamics of ligand-TXAS interactions by steady-state and time-resolved fluorescence spectroscopy. The proximity between TNS and each of the five tryptophan (Trp) residues in TXAS was examined through the fluorescence quenching of Trp by TNS via an energy transfer process. The fluorescence quenching of Trp by TNS was abolished in the W65F mutant, indicating that Trp65 is the major contributor to account for energy transfer with TNS. Furthermore, both competitive binding experiments and the computer-simulated TXAS structure with clotrimazole as a heme ligand strongly suggest that TXAS has a large active site that can simultaneously accommodate TNS and clotrimazole without mutual interaction between TNS and heme. Displacement of TNS by Nile Red, a fluorescence dye sensitive to environmental polarity, indicates that the TNS binding site in TXAS is likely to be hydrophobic. The Phe cluster packing near the binding site of TNS may be involved in facilitating the binding of multiple ligands to the large active site of TXAS.

Functional analysis of human thromboxane synthase polymorphic variants.

BACKGROUND: Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane H2 (TXA2), a potent inducer of blood vessel constriction and platelet aggregation. Nonsynonymous polymorphisms in the TXAS gene have the potential to alter TXAS activity and affect TXA2 generation. OBJECTIVES: The aim of this study was to assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K, and T451N. METHODS: Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA2 and fragmentation of PGH2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. RESULTS: All of the variants showed both isomerization and fragmentation activities. The Km values of the variants ranged from 27 to 52 µmol/l PGH2 (wild-type value: 32 µmol/l PGH2); the Vmax values of the variants ranged from 18 to 40 U/mg (wild-type value: 41 U/mg). The kinetic differences were largest for the L357V variant, whose Vmax/Km ratio was just 27% of the wild-type value. CONCLUSION: The increased Km and decreased Vmax values observed with L357V suggest that this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.

Genome wide analysis and comparative docking studies of new diaryl furan derivatives against human cyclooxygenase-2, lipoxygenase, thromboxane synthase and prostacyclin synthase enzymes involved in inflammatory pathway.

In an effort to develop potent anti-inflammatory and antithrombotic drugs, a series of new 4-(2-phenyltetrahydrofuran-3-yl) benzene sulfonamide analogs were designed and docked against homology models of human cyclooxygenase-2 (COX-2), lipoxygenase and thromboxane synthase enzymes built using MODELLER 7v7 software and refined by molecular dynamics for 2 ns in a solvated layer. Validation of these homology models by procheck, verify-3D and ERRAT programs revealed that these models are highly reliable. Docking studies of 4-(2-phenyltetrahydrofuran-3-yl) benzene sulfonamide analogs designed by substituting different chemical groups on benzene rings replacing 1H pyrazole in celecoxib with five membered thiophene, furan, 1H pyrrole, 1H imidazole, thiazole and 1,3-oxazole showed that diaryl furan molecules showed good binding affinity towards mouse COX-2. Further, docking studies of diaryl furan derivatives are likely to have superior thromboxane synthase and COX-2 selectivity. Docking studies against site directed mutagenesis of Arg120Ala, Ser530Ala, Ser530Met and Tyr355Phe enzymes displayed the effect of inhibition of COX-2. Drug likeliness and activity decay for these inhibitors showed that these molecules act as best drugs at very low concentrations.

SAR index: quantifying the nature of structure-activity relationships.

Structure-activity relationships (SARs) can display very different features. Small chemical modifications of active molecules often dramatically alter biological responses. By contrast, structurally diverse molecules can have similar activity. SARs can also be heterogeneous in nature. For example, for structurally diverse molecules with similar activity, closely related analogs might have significant differences in potency. Given the inherent complexity of SARs, it has been very difficult to estimate SAR characteristics from molecular structure. On the basis of systematic correlation of 2D structural similarity and compound potency, we have developed a function termed "SAR Index" that quantitatively describes the nature of SARs and establishes different SAR categories: continuous, discontinuous, heterogeneous-relaxed, and heterogeneous-constrained. These heterogeneous SAR categories are described for the first time. Given a set of active compounds and their potency values, SAR Index calculations can estimate how likely it is to identify structurally distinct molecules having similar activity.

Solution structure of a common substrate mimetic of cyclooxygenase-downstream synthases bound to an engineered thromboxane A2 synthase using a high-resolution NMR technique.

Understanding the docking mechanism of the common substrate, prostaglandin H(2) (PGH(2)), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH(2) to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH(2) analogue, bound to one of the COX-downstream synthases-an engineered thromboxane A(2) synthase (TXAS). The dynamic binding was clearly observed by (1)D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D (1)H NMR experiments using transferred NOEs. Through the assignments for the 2D (1)H NMR spectra, TOCSY, DQF-COSY, NOESY, and the structural calculations based on the NOE constraints, they demonstrated that the widely open conformation with a triangle shape of the free U44069 changed to a compact structure with an oval shape when bound to the TXAS. The putative substrate-binding pocket of the TXAS model fits the conformation of the TXAS-bound U44069 appropriately, but could not fit the free form of U44069. It was the first to provide structural information for the dynamic docking of the PGH(2) mimic of the TXAS in solution, and to imply that PGH(2) undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH(2) to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH(2) bound to other COX-downstream synthases.

Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form.

Thromboxane A2 synthase (TXAS) binds to the endoplasmic reticulum membrane and catalyzes both an isomerization of prostaglandin H2 (PGH2) to form thromboxane A2 (TXA2) and a fragmentation of PGH2 to form 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA). TXAS is a non-classic cytochrome P450 in that it does not require molecular oxygen or an external electron donor for catalysis. Difficulty in obtaining crystals from the membrane-bound TXAS prompted us to modify the protein to a soluble form. Results from site-directed mutagenesis, hydropathy analysis, and homology modeling led us to identify a putative membrane association segment near the end of helix F in TXAS. We report here the generation of a soluble form of TXAS by deletion of the amino-terminal membrane-anchoring domain and replacement of the helix F and F-G loop region with the corresponding region of the structurally characterized microsomal P450 2C5. The resultant TXAS/2C5 chimera is expressed in bacteria as a cytosolic and monomeric protein. Addition of an amino-terminal leader sequence to enhance expression and a tetra-histidine segment at the carboxyl-terminus to facilitate purification yielded approximately 4 mg of nearly homogeneous TXAS/2C5 per liter of bacterial culture. The TXAS/2C5 chimera contains heme at nearly a 1:1 molar ratio and catalyzes the formation of TXA2, MDA, and HHT at a 1:1:1 ratio, although with a reduced catalytic activity compared to wild type TXAS. TXAS/2C5 exhibits electronic absorption spectra similar to wild type TXAS and has similar affinities toward distal heme ligands such as imidazole and U44069. The chimera was mono-dispersive and thus is promising for crystallization trials.

Resonance Raman investigation of the interaction of thromboxane synthase with substrate analogues.

Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.

Thoughts on thiolate tethering. Tribute and thanks to a teacher.

A unique feature of P450 enzymes is in the presence of a thiolate ligand heme but its exact function in catalysis is a matter of debate. For P450 dependent monooxygenases the "active oxygen" complex seems to exist only as a transition state in which the thiolate ligand provides electron density in order to prevent pi-backbonding of the oxygen to the iron (-S-Fe-O(z.rad;)). The corresponding ground state (Compound I) would be a ferryl species (Fe(IV)z.dbnd6;O) with an electron hole either at the porphyrin or at the sulfur. Apart from this role we postulate that a second function is related to the electronic structure of Compound II as an electron acceptor and this property is shared among monooxygenases, thromboxane synthase, prostacyclin synthase, allene oxide synthase, P450(NOR(-)) and chloroperoxidase. As a common step in all P450 enzymes an extremely rapid electron uptake by Compound II allows that the primary substrate radicals are oxidized to cations which immediately combine with a neighbouring nucleophile. Thus "electron transfer" may substitute for "oxygen rebound" as the final step leading to product formation. The same principle also applies methane monooxygenases in which the role of the thiyl sulfur is replaced by a ferryl-oxyl entity.

Novel agents combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI).

Compounds 1 or 2 which possess dual-acting PAF antagonist/TxSI in a previous paper were modified and evaluated for the dual-acting activity. It was found that several compounds were potent dual-acting PAF antagonist/TxSI in and ex vivo. 6-(2-Chlorophenyl)-3-[4-[(E/Z)-6-ethoxycarbonyl-1-(3-pyridyl)-1-hexenyl]phenylmethyl]-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3': 4,5]thieno[3,2-f]triazolo[4,3-a]diazepine (12) is excellent orally dual-acting PAF antagonist/TxSI.

Approach to dual-acting platelet activating factor (PAF) receptor antagonist/thromboxane synthase inhibitor (TxSI) based on the link of PAF antagonists and TxSIs.

A series of compounds (22-36) which possess dual-acting PAF antagonist/TxSI have been generated by the approach of linking the known PAF antagonists and TxSIs, such as Ridogrel (1).

Identification of the substrate interaction site in the N-terminal membrane anchor segment of thromboxane A2 synthase by determination of its substrate analog conformational changes using high resolution NMR technique.

The present studies describe an investigation for the interaction of N-terminal membrane anchor domain of thromboxane A(2) synthase (TXAS) with its substrate analog in a membrane-bound environment using the two-dimensional NMR technique. TXAS and prostaglandin I(2) synthase (PGIS), respectively, convert the same substrate, prostaglandin H(2) (PGH(2)), to thromboxane A(2) and prostaglandin I(2), which have opposite biological functions. Our topology studies have indicated that the N-terminal region of TXAS has a longer N-terminal endoplasmic reticulum (ER) membrane anchor region compared with the same segment proposed for PGIS. The differences in their interaction with the ER membrane may have an important impact to facilitate their common substrate, PGH(2), across the membrane into their active sites from the luminal to the cytoplasmic side of the ER. To test this hypothesis, we first investigated the interaction of the TXAS N-terminal membrane anchor domain with its substrate analog. A synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1-35) of TXAS, which adopted a stable helical structure and exhibited a membrane anchor function in the membrane-bound environment, was used to interact with a stable PGH(2) analog,. High resolution two-dimensional NMR experiments, NOESY and TOCSY, were performed to solve the solution structures of in a membrane-mimicking environment using dodecylphosphocholine micelles. Different conformations were clearly observed in the presence and absence of the TXAS N-terminal membrane anchor domain. Through combination of the two-dimensional NMR experiments, completed (1)H NMR assignments of were obtained, and the data were used to construct three-dimensional structures of in H(2)O and dodecylphosphocholine micelles, showing the detailed conformation change upon the interaction with the membrane anchor domain. The observation supported the presence of a substrate interaction site in the N-terminal region. The combination of the structural information of and was able to simulate a solution structure of the unstable TXAS and PGIS substrate, PGH(2).

Structural characterization and topology of the second potential membrane anchor region in the thromboxane A2 synthase amino-terminal domain.

Thromboxane A2 synthase (TXAS) has been proposed to have two membrane-bound regions located in the NH2-terminal domain [Ruan, K.-H., Wang, L.-H., Wu, K. K., and Kulmacz, R. J. (1993) J. Biol. Chem, 268, 19483-19489; Ruan, K.-H., Li, P., Kulmacz, J. R., and Wu, K. K. (1994) J. Biol. Chem, 269, 20938-20942]. To test this hypothesis, a solution structure in membrane mimetic environments of a synthetic peptide corresponding to the second region of the NH2-terminal domain (TXAS residues 33-60) has been investigated by circular dichroism (CD), 2D nuclear magnetic resonance (NMR) spectroscopy, and peptidoliposome reconstitution. CD spectroscopy indicated that the peptide adopted a structure with significant alpha-helical content in 30% trifluoroethanol (TFE) or in dodecylphosphocholine (DPC) micelles, which mimic hydrophobic membrane environment. Through a combination of 2D NMR experiments in the presence of TFE or DPC micelles, complete 1H NMR assignments of the peptide have been obtained and the structure of the peptide has been determined. NH2-terminal segment of the peptide takes on a well-defined alpha-helical conformation; the center segment of the peptide, containing three prolines, adopts a bent conformation, and the C-terminal segment of the peptide exists in a mixture of rapidly interconverting conformations. These results provide direct structural evidence that residues 33-60 of the TXAS NH2-terminal domain contain a second membrane anchor region, with at least residues 35-46 having their helical structure expected for hydrophobic interaction with the membrane. The orientation of the peptide in DPC micelles was evaluated from the effect of incorporation of a spin-label 12-doxylstearate into the micelles. The peptide portions, found to be immersed in the micelles, include the helical segment, the bent segment, and some hydrophobic residues within the C-terminal segment. Two additional synthetic peptides, one corresponding to the NH2-terminal helical segment (TXAS residues 33-46) and the other including the bent and the C-terminal segments (TXAS residues 47-60) were analyzed for their ability to incorporate into peptidoliposomes. The helical peptide readily incorporated into liposomes; the other peptide did not. These results support the presence of a second functional membrane anchor region localized to the helical segment within TXAS residues 33-46, with passive membrane contacts in the bent and the C-terminal segments of the peptide (TXAS residues 47-60) due to immersion of the helical in the membrane.

Immunoreactive thromboxane synthase is measurable in ovine fetal hypothalamus as early as 86 days' gestation.

Thromboxane A2 (TxA2) augments hypothalamus-pituitary-adrenal axis activity in both fetal and adult animals. We have proposed that TxA2 acts as a neuromodulator within the brain to stimulate the release of corticotropin releasing hormone (CRH) or arginine vasopressin (AVP) into the hypophyseal-portal blood. We performed the present experiments to identify immunoreactive thromboxane synthase (TxS) within fetal brain regions and to quantify developmental changes in the TxS immunoreactivity measurable within those regions. We found that immunoreactive TxS was present in fetal hypothalamus, pituitary, brainstem, and lung. In fetal hypothalamus, we found immunoreactive TxS in three identifiable molecular weights, approximately 65, 42, and 35 kD. In fetal pituitary and lung, we found the 65 and 35 kD forms, and in the brainstem we found only the 35 kD form. In fetal pituitary, there was a clear ontogenetic change in TxS immunoreactivity. The 42 kD TxS immunoreactivity was not present in the youngest fetal sheep studied (86-90 days' gestation), but was expressed in the other age groups (125-128, 135-139, 141-term, and postnatal ages). The other molecular weight forms appeared to increase in the older fetuses, but the changes were not significant. In the hypothalamus, all three forms of TxS were measurable at all ages, and there was no significant change in relative abundance. We conclude that immunoreactive TxS is present in the fetal brain throughout the last half of fetal gestation, but that the significance of multiple molecular weight forms is not clear.