RESUMEN
Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Aspirina/farmacología , Proteínas Portadoras/metabolismo , Fibroblastos/efectos de los fármacos , Proteínas de Choque Térmico , Chaperonas Moleculares/metabolismo , Salicilato de Sodio/farmacología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Aspirina/metabolismo , Unión Competitiva/efectos de los fármacos , Radioisótopos de Carbono , Sistema Libre de Células/metabolismo , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Masculino , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas/aislamiento & purificación , Proteínas/metabolismoRESUMEN
Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.
Asunto(s)
Fragmentos de Péptidos/química , Receptores de Tromboxanos/química , Tromboxano A2/metabolismo , Dicroismo Circular , Espacio Extracelular/química , Humanos , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Tromboxanos/metabolismo , Soluciones , Relación Estructura-ActividadRESUMEN
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).
Asunto(s)
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/química , Endoperóxidos de Prostaglandinas Sintéticos/química , Tromboxano-A Sintasa/química , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Sitios de Unión , Espectroscopía de Resonancia Magnética , Conformación Molecular , Endoperóxidos de Prostaglandinas Sintéticos/metabolismo , Relación Estructura-Actividad , Tromboxano-A Sintasa/fisiologíaRESUMEN
Prostaglandin I(2) synthase (PGIS) is an eicosanoid-synthesizing cytochrome P450, located in the endoplasmic reticulum (ER) membrane. The membrane topology of the catalytic portion of PGIS is still unknown. General models of the membrane topology of microsomal P450s have been proposed in two forms: (a) large part of the polypeptide exposed on the cytoplasmic side with an NH(2)-terminal membrane anchor to the ER membrane and (b) deep immersion of the polypeptide in the membrane, as described by J. P. Miller et al. (1996, Biochemistry 35, 1466-1474). We have characterized the membrane topology of catalytic portion of PGIS using molecular modeling-guided site-specific antibodies. A 3D working model of PGIS was constructed by homology modeling using P450(BM-3) crystal structure as a template (S. K. Shyue et al., 1997, J. Biol. Chem. 272, 3657-3662). Three hydrophilic peptides corresponding to different regions of the surface portion of PGIS with residues 109-127 (P109-127), 353-368 (P353-368), and 411-431 (P411-431) predicted from the model and an NH(2)-terminal hydrophobic peptide (residues 1-28, P1-28) were synthesized and used to prepare site-specific antibodies. All three of the hydrophilic peptide antibodies have high titer and are specifically recognized human PGIS, as shown by binding assays and Western blot analysis. In contrast, the hydrophobic NH(2)-terminal peptide has a much lower titer binding to the PGIS protein. The overall arrangement of the PGIS polypeptide with respect to the endoplasmic reticulum (ER) membrane was examined by immunocytochemistry techniques in transiently transfected COS-1 cells with recombinant human PGIS cDNA and in ECV cells expressing endogenous PGIS. The immunofluorescence staining for the cells with selective permeabilization of the plasma membrane using streptolysin O indicated that all three of the hydrophilic peptide antibodies bound to the cytoplasmic surface of the ER membrane. These results provide direct experimental evidence supporting the predicted 3D protein topological model in which the segments are located on the protein surface and the membrane topological model in which PGIS is largely exposed on the cytoplasmic side of the ER membrane. It also led us to conclude that the PGIS substrate, prostaglandin H(2) (PGH(2)), produced by prostaglandin H(2) synthase (PGHS) in the ER lumenal side must pass through the ER membrane barrier to the catalytic site of the PGIS in the cytoplasmic side of the ER membrane.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/inmunología , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/inmunología , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Línea Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Prostaglandina H2 , Prostaglandinas H/metabolismo , Conformación Proteica , TransfecciónRESUMEN
Prostaglandin I2 synthase (PGIS) produces prostaglandin I2 (PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2 (TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2 synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Oxidorreductasas Intramoleculares/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , Eicosanoides/biosíntesis , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Liposomas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , ConejosRESUMEN
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.
Asunto(s)
Proteínas de la Membrana/química , Estructura Terciaria de Proteína , Tromboxano-A Sintasa/química , Secuencia de Aminoácidos , Dicroismo Circular , Liposomas/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/metabolismo , Micelas , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Relación Estructura-Actividad , Tromboxano-A Sintasa/metabolismoRESUMEN
Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450BM-3 crystal structure as the template. Our results indicate that residues Ile67, Val76, Leu384, Pro355, Glu360, and Asp364, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing beta1-1, beta1-2, beta1-3, and beta1-4 strands is predicted well by the model. At the heme region, Cys441 was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450BM-3 were substituted by Leu112 and Asp439, respectively in PGIS. Alteration of Leu112 to Phe retained the activity, indicating that Leu112 is a functional substitution for Phe. In contrast, mutant Asp439 --> Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Oxidorreductasas Intramoleculares , Isomerasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Células COS , Cromatografía en Capa Delgada , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de SecuenciaAsunto(s)
Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , Isoenzimas/biosíntesis , Isoenzimas/química , Microsomas/enzimología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/química , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Células COS , Ciclooxigenasa 1 , Retículo Endoplásmico/ultraestructura , Epítopos/análisis , Humanos , Membranas Intracelulares/ultraestructura , Proteínas de la Membrana , Microsomas/ultraestructura , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Ovinos , TransfecciónRESUMEN
Prostaglandin H synthase (PGHS), a key enzyme in prostanoid biosynthesis, exists as two isoforms. PGHS-1 is considered a basal enzyme; PGHS-2 is associated with inflammation and cell proliferation. A number of highly selective inhibitors for PGHS-2 cyclooxygenase activity are known. Inhibition by these agents involves an initial reversible binding, followed by a time-dependent transition to a much higher affinity enzyme-inhibitor complex, making these agents potent and poorly reversible PGHS-2 inhibitors. To investigate the PGHS-2 structural features that influence the time-dependent action of the selective inhibitors, we have constructed a three-dimensional model of human PGHS-2 by homologous modeling. Examination of the PGHS-2 model identified Val509 as a cyclooxygenase active site residue, that was not conserved in PGHS-1. Recombinant human PGHS-2 with Val509 mutated to either Ile (the corresponding residue in PGHS-1), Ala, Glu, or Lys was expressed by transient transfection of COS-1 cells to evaluate the effects of the mutations on cyclooxygenase activity and on inhibition by four agents reported to be selective for PGHS-2 (NS398, nimesulide, DuP697, and SC58125). All the recombinant proteins were of the expected mass. The mutants exhibited 45-210% of wild-type cyclooxygenase activity, with Km values for arachidonate of 2.1-7.6 microM (wild-type PGHS-2, 3.8 microM), indicating that changes in position 509 had modest effects on cyclooxygenase catalysis. Each of the agents inhibited wild-type PGHS-2 in a time-dependent fashion, and all but nimesulide did the same for the V509A mutant. In contrast, the V509E and V509I PGHS-2 mutants, like recombinant human PGHS-1, did not show time-dependent inhibition with any of the agents, and the V509K mutant responded in a time-dependent manner only to DuP697. Reversible inhibition was still observed with Val509 mutants that did not show time-dependent inhibition. Thus, the side chain structure at position 509 markedly influenced the ability of PGHS-2 to undergo the time-dependent transition without removing inhibitor or substrate binding. These results indicate that Val509 in PGHS-2 has a major role in the structural transition that underlies time-dependent inhibition by the isoform-selective agents.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/antagonistas & inhibidores , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Conejos , Valina/metabolismoRESUMEN
Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structure-function relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting that oxygen bond cleavage is not a rate-limiting step in thromboxane A2 biosynthesis. The present results from site-directed mutagenesis support the overall structure of the TXAS active site predicted by homology modeling and have allowed refinement of the position of bound substrate.
Asunto(s)
Tromboxano-A Sintasa/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Simulación por Computador , Sistema Enzimático del Citocromo P-450/química , Cartilla de ADN/química , Humanos , Cinética , Microsomas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Prostaglandina H2 , Prostaglandinas H/metabolismo , Relación Estructura-Actividad , Tromboxano-A Sintasa/metabolismoRESUMEN
Prostaglandin H synthase-1 is an integral endoplasmic reticulum membrane protein which catalyzes a key control step in prostaglandin biosynthesis. The overall arrangement of the prostaglandin H synthase-1 polypeptide with respect to the endoplasmic reticulum membrane was examined in transiently transfected COS-1 cells, using immunofluorescence microscopy. A bacterial toxin, streptolysin-O, was used for selective plasma membrane permeabilization and a detergent, saponin, for general membrane permeabilization. Treated cells were probed with six antibodies specific for particular prostaglandin H synthase-1 peptide segments and one antibody specific for an inserted viral reporter epitope. Control experiments established that actin, a cytoplasmic marker, was accessible to fluorescein-labeled phalloidin after streptolysin-O treatment, whereas antibodies against protein disulfide isomerase, an endoplasmic reticulum lumenal marker, bound only after saponin treatment, Using this approach to investigate prostaglandin H synthase-1, it was found that streptolysin-O treatment was sufficient to obtain staining of intracellular membranes by antibodies specific for the endogenous C-terminal segment, for the viral reporter inserted at the C-terminus, and for the protease-sensitive region near arg277. In contrast, saponin treatment was necessary for staining by antibodies specific for peptides spanning residues 51-66, 156-170, and 377-390. Antibodies targeted against residues 483-496 did not stain transfected cells even after saponin permeabilization, although they did bind to detergent-solubilized prostaglandin H synthase-1. These results indicate that the C-terminus and arg277 regions of the synthase can be exposed on the cytoplasmic side of the endoplasmic reticulum membrane, whereas regions near N-glycosylation sites are confined to the endoplasmic reticulum lumen and residues 483-496 are inaccessible from either side of the endoplasmic reticulum membrane.
Asunto(s)
Retículo Endoplásmico/enzimología , Prostaglandina-Endoperóxido Sintasas/análisis , Secuencia de Aminoácidos , Anticuerpos , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/inmunología , TransfecciónAsunto(s)
Retículo Endoplásmico/enzimología , Prostaglandina-Endoperóxido Sintasas/química , Animales , Sitios de Unión de Anticuerpos , Línea Celular , Chlorocebus aethiops , Humanos , Riñón , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Mutación Puntual , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , TransfecciónRESUMEN
A 3-D model of human thromboxane A2 synthase (TXAS) was constructed using a homology modeling approach based on information from the 2.0 A crystal structure of the hemoprotein domains of cytochrome P450BM-3 and P450cam. P450BM-3 is a bacterial fatty acid monooxygenase resembling eukaryotic microsomal cytochrome P450s in primary structure and function. TXAS shares 26.4% residue identity and 48.4% residue similarity with the P450BM-3 hemoprotein domain. The homology score between TXAS and P450BM-3 is much higher than that between TXAS and P450cam. Alignment between TXAS and the P450BM-3 hemoprotein domain or P450cam was determined through sequence searches. The P450BM-3 or P450cam main-chain coordinates were applied to the TXAS main chain in those segments where the two sequences were well aligned. These segments were linked to one another using a fragment search method, and the side chains were added to produce a 3-D model for TXAS. A TXAS substrate, prostaglandin H2 (PGH2) was docked into the TXAS cavity corresponding to the arachidonic acid binding pocket in P450BM-3 or camphor binding site in P450cam. Regions of the heme and putative PGH2 binding cavities in the TXAS model were identified and analyzed. The segments and residues involved in the active-site pocket of the TXAS model provide reasonable candidates for TXAS protein engineering and inhibitor design. Comparison of the TXAS model based on P450BM-3 with another TXAS model based on the P450cam structure indicated that P450BM-3 is a more suitable template for homology modeling of TXAS.
Asunto(s)
Proteínas Bacterianas , Tromboxano-A Sintasa/química , Secuencia de Aminoácidos , Sitios de Unión , Alcanfor 5-Monooxigenasa , Simulación por Computador , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/química , Hemo/química , Humanos , Oxigenasas de Función Mixta/química , Modelos Moleculares , Datos de Secuencia Molecular , NADPH-Ferrihemoproteína Reductasa , Prostaglandina H2 , Prostaglandinas H/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Thromboxane A2 synthase (TXAS), a member of the cytochrome P450 superfamily, is believed to be anchored to the endoplasmic reticulum membrane by hydrophobic portions of its NH2-terminal domain. Two hydrophobic peptides, corresponding to potential membrane-anchor segments of the NH2-terminal region of TXAS (residues 1-36, designated LP1 and residues 33-60, designated LP2) were synthesized, and their secondary structure and ability to insert in a lipid bilayer were characterized. The conformation of the synthetic peptides were analyzed in organic and membrane (bilayer) environments. Circular dichroism spectroscopy indicated that both segments adopt structures with significant alpha-helical content in a hydrophobic environment. For the LP1 peptide, the helical content was maximal with 60% trifluoroethanol, whereas 20% trifluoroethanol maximized the helix content for the LP2 peptide. The interaction of several NH2-terminal peptides with a lipid bilayer was determined by reconstitution of these peptides into lipid vesicles composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Both the LP1 and LP2 peptides bound strongly to the defined lipid vesicles, but peptides corresponding to residues 1-15 and 33-36 were not incorporated into the lipid vesicles. The results indicate that TXAS has two distinct membrane-anchor segments, comprising residues 16-33 and 37-60 in its NH2-terminal domain.
Asunto(s)
Tromboxano-A Sintasa/química , Tromboxano-A Sintasa/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Cromatografía en Gel , Dicroismo Circular , Liposomas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Secundaria de ProteínaRESUMEN
The membrane topology of the NH2-terminal portion of human thromboxane synthase (TXS), a member of the cytochrome P450 superfamily, has been investigated. By sequence alignment, the first 6 residues of the mature TXS polypeptide are likely to form a distinctive "tail" structure not found in many other mammalian cytochromes P450 in the endoplasmic reticulum membrane. Peptides with either the ultimate 10 or 15 residues of the NH2 terminus of TXS were synthesized and used to produce site-directed antibodies. The resulting peptide antibodies were highly specific and recognized human TXS, as shown by binding assays and Western blot analysis. Binding of the peptide antibodies to recombinant TXS in transfected COS-1 and to endogenous TXS in THP-1 cells was analyzed by immunocytochemistry. Selective permeabilization of the plasma membrane to immunoglobulin was achieved with streptolysin O; general permeabilization, including the endoplasmic reticulum membrane, was accomplished with Triton X-100. Permeabilization of the plasma membrane was sufficient to produce binding of both peptide antibodies to their epitopes, indicating that the epitopes for both of the peptide antibodies were exposed on the cytoplasmic side of the endoplasmic reticulum membrane. The results with the peptide antibodies provide direct experimental evidence supporting the topological model for membrane-bound cytochrome P450 proposed by Nelson and Strobel (Nelson, D. R., and Strobel, H. W. (1988) J. Biol. Chem. 263, 6038-6050), in which the NH2 terminus is oriented toward the cytoplasmic side of the endoplasmic reticulum membrane.
Asunto(s)
Plaquetas/enzimología , Retículo Endoplásmico/enzimología , Microsomas/enzimología , Tromboxano-A Sintasa/química , Tromboxano-A Sintasa/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Secuencia de Bases , Sitios de Unión de Anticuerpos , Western Blotting , Línea Celular , Clonación Molecular , Retículo Endoplásmico/ultraestructura , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Humanos , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tromboxano-A Sintasa/sangre , TransfecciónRESUMEN
A highly sensitive fluorimetric enzyme immunoassay was developed for prostaglandin H (PGH) synthase, an intrinsic membrane protein, using n-octyl beta-D-glucopyranoside for solubilization of the synthase from the endoplasmic reticulum membrane. An anti-PGH synthase IgG-coated polystyrene ball was reacted with a PGH synthase standard or crude detergent extract of cells, washed to remove the detergent, and then incubated with anti-PGH synthase Fab'-beta-D-galactosidase conjugate. The bound beta-D-galactosidase activity was quantitated with 4-methylumbelliferyl-beta-D-galactoside, a fluorogenic substrate. The detection limit for the PGH synthase standard was found to be 1.0 pg/assay. The sensitivity of this assay is increased by about 2-3 orders of magnitude over those of previously reported radioimmunoassay or colorimetric enzyme immunoassay. The high sensitivity of the fluorimetric enzyme immunoassay allowed reliable detection of low levels of human PGH synthase present in small samples of cultured cells. Our studies with this fluorimetric immunoassay for the synthase developed a general method for determination of membrane-bound proteins at ultrasensitive levels.
Asunto(s)
Fluorometría/métodos , Técnicas para Inmunoenzimas , Prostaglandina-Endoperóxido Sintasas/análisis , Animales , Especificidad de Anticuerpos , Células Cultivadas/enzimología , Endotelio Vascular/enzimología , Glucósidos , Glicósidos , Humanos , Himecromona/análogos & derivados , Proteínas de la Membrana/análisis , Prostaglandina-Endoperóxido Sintasas/aislamiento & purificación , Ovinos , beta-Galactosidasa/análisisRESUMEN
A retroviral vector (BAG) was used to transfer human prostaglandin H synthase (PGHS-1) gene into a human endothelial cell line for enhancement of PGI2 synthesis. Cells infected with BAG containing PGHS-1 cDNA in the sense orientation relative to the retroviral promoter (PGHS(S)) expressed a 30-fold increase in mRNA but, due to a reading frame shift, did not show an increase in PGHS protein or in PGI2 synthesis, while those with PGHS-1 in reverse orientation relative to the viral promoter (PGHS(R)), produced a > 10-fold increase in PGHS mRNA over the control (169 +/- 22 vs 14.8 +/- 1.2 amol/micrograms RNA) with a concordant increase in PGHS protein (5.82 +/- 1.07 vs 0.23 +/- 0.04 ng/mg protein) and enzyme activity. Primer extension analysis of PGHS(R) revealed two transcription start sites located in the SV40 late promoter region adjacent to PGHS-1 cDNA. PGHS(R) cells produced a high basal PGI2 level which was increased by several-fold in response to stimulation by ionophore, arachidonic acid, and thrombin. Kinetic analysis revealed the PGI2 synthetic rate to be 14 ng/min-1 per million cells and t1/2 of PGI2 synthesis, 13.3 min. These findings indicate that transfer of PGHS-1 gene into vascular cells enhances PGI2 synthesis and may be a useful strategy for restoring thromboprotective property of damaged blood vessels.
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ADN/genética , Endotelio Vascular/enzimología , Epoprostenol/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Retroviridae/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Línea Celular , Endotelio Vascular/citología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transfección/fisiologíaRESUMEN
Eighteen synthetic overlapping peptides encompassing the entire extracellular part (residues alpha 1-210) of the alpha-chain of human acetylcholine receptor (AChR) and a 19th peptide (residues alpha 262-276) corresponding to an extracellular connection between two transmembrane regions were prepared and used for the measurement, by solid-phase radioimmunoassay, of the binding of autoantibodies in plasma from myasthenia gravis (MG) patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides. The regions recognized resided predominantly within the areas alpha 10-30, alpha 111-145 and alpha 175-198 and, less frequently, region alpha 45-77. Differences in the recognition profile of the peptides from patient to patient indicated that the autoantibody responses were under genetic control. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 myasthenia sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases. The mapping of the continuous antigenic regions recognized by autoantibodies on the alpha-chain of human AChR has permitted a comparison of the regions recognized by autoantibodies and autoimmune T-cells from the same donor. It also provided a peptide-based direct antibody binding method for diagnosis of MG.
Asunto(s)
Autoanticuerpos/metabolismo , Epítopos/química , Miastenia Gravis/inmunología , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Ciclofosfamida/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Miastenia Gravis/diagnóstico , Miastenia Gravis/tratamiento farmacológico , Fragmentos de Péptidos/metabolismo , Prednisona/uso terapéutico , Bromuro de Piridostigmina/uso terapéutico , Radioinmunoensayo , Linfocitos T/inmunologíaRESUMEN
To determine alpha-fetoprotein (AFP) in human saliva, a highly sensitive sandwich enzyme immunoassay for saliva AFP was developed. AFP standards and saliva samples were added into the wells of a polystyrene plate coated with goat IgG antibody against human AFP. After incubation, the wells were washed and horseradish peroxidase-labelled antibody was added. The enzyme activity specifically bound to the well was assayed using 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide as substrate. The reaction was stopped by addition of 2 M sulphuric acid and the AFP concentration was determined from the absorbance at 450 nm. The minimum detectable concentration was 8 ng/L. The recovery of AFP mixed with human saliva was 91.1-102.4%. The within-assay and between-assay coefficients of variation were 6.5-8.9% and 7.6-10.8%, respectively. The assay correlated well with a radioimmunoassay for human AFP (r = 0.985, n = 13, P less than 0.001). The mean concentration of AFP in normal human saliva was 14.3 ng/L (SEM = 4.9 ng/L, n = 10) and significantly higher levels of saliva AFP were observed in hepatocellular carcinoma patients with positive serum AFP (mean 1367.8 ng/L, SEM 595.4 ng/L, n = 6; P less than 0.001). Strong correlation was observed between saliva AFP and serum AFP (r = 0.978, P less than 0.01, n = 13).
Asunto(s)
Carcinoma Hepatocelular/química , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/química , Saliva/química , alfa-Fetoproteínas/análisis , Adolescente , Adulto , Interpretación Estadística de Datos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Radioinmunoensayo , Distribución Aleatoria , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A synthetic peptide corresponding to a myasthenogenic region of Torpedo californica acetylcholine (AcCho) receptor (AcChoR) alpha subunit, AcChoR alpha-(125-148), was conjugated to monomethoxypolyethylene glycol (mPEG). Injection of mice with the mPEG-AcChoR alpha-(125-148) conjugate and subsequent immunization with whole Torpedo AcChoR suppressed the development of experimental autoimmune myasthenia gravis (EAMG) by electrophysiological criteria. In anti-AcChoR sera from these animals, the antibody response against unconjugated AcChoR alpha-(125-148) was decreased, while the antibody responses against whole AcChoR and other epitopes were not altered. There were no detectable changes in T-cell proliferation responses to AcChoR alpha-(125-148) or to whole AcChoR in these animals. Prior injections with a "nonsense" peptide-mPEG conjugate had no effect on responses to the subsequent immunization with whole Torpedo AcChoR. The results indicate that the mPEG-AcChoR alpha-(125-148) conjugate has epitope-specific tolerogenicity for antibody responses in EAMG and that the AcChoR alpha-subunit region comprising residues 125-148 plays an important pathophysiological role in EAMG. The epitope-directed tolerogenic conjugates may be useful for future immunotherapies of human myasthenia gravis. The strategy of specific suppression of the antibody response to a predetermined epitope by using a synthetic mPEG-peptide conjugate may prove useful in manipulation and suppression of unwanted immune responses such as autoimmunity and allergy.