RESUMEN
Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Fluoresceínas/química , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Línea Celular , Interacciones Farmacológicas , Humanos , Sensibilidad y EspecificidadRESUMEN
Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Antihelmínticos/farmacología , Lactonas/farmacología , Compuestos Macrocíclicos/farmacología , Proteínas de Neoplasias/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Absorción , Animales , Resistencia a Múltiples Medicamentos , Humanos , Ivermectina/farmacología , Lípidos/química , Proteínas de Neoplasias/genéticaRESUMEN
Multidrug resistance protein 2 (MRP2) is a multispecific organic anion transporter expressed at important pharmacological barriers, including the canalicular membrane of hepatocytes. At this location it is involved in the elimination of both endogenous and exogenous waste products, mostly as conjugates, to the bile. Estradiol-17beta-d-glucuronide (E(2)17betaG), a widely studied endogenous substrate of MRP2, was shown earlier to recognize two binding sites of the transporter in vesicular transport assays. MRP2 modulators (substrates and nonsubstrates) potentiate the transport of E(2)17betaG by MRP2. We correlated data obtained from studies of different complexities and investigated the species-specific differences between rat and human MRP2-mediated transport. We used vesicular transport assays, sandwich-cultured primary hepatocytes, and in vivo biliary efflux in rats. Our results demonstrate that the rat Mrp2 transporter, unlike the human MRP2, transports E(2)17betaG according to Michaelis-Menten type kinetics. Nevertheless, in the presence of modulator drugs E(2)17betaG transport mediated by the rat transporter also shows cooperative kinetics as potentiation of E(2)17betaG transport was observed in the vesicular transport assay. We also demonstrated that the potentiation exists both in rat and in human hepatocytes and in vivo in rats.
Asunto(s)
Estradiol/análogos & derivados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Animales , Transporte Biológico , Células Cultivadas , Estradiol/metabolismo , Estradiol/farmacocinética , Hepatocitos/metabolismo , Humanos , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
BACKGROUND: Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs. OBJECTIVES: To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation of the interactions. METHODS: Different in vitro membrane-based methods (ATPase and vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and cytotoxicity assay) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP. RESULTS: In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown by the vanadate-sensitive ATPase assay. In line with the membrane assays, leflunomide and A771726 inhibited BCRP-mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay, overexpression of BCRP conferred 20.6-fold and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP. CONCLUSION: Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Compuestos de Anilina/farmacología , Antirreumáticos/farmacología , Hidroxibutiratos/farmacología , Isoxazoles/farmacología , Proteínas de Neoplasias/efectos de los fármacos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adenosina Trifosfatasas/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Crotonatos , Resistencia a Medicamentos , Humanos , Leflunamida , Nitrilos , ToluidinasRESUMEN
ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Membrana Celular/metabolismo , Colesterol/fisiología , Proteínas de Neoplasias/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/metabolismo , Animales , Baculoviridae/genética , Bencimidazoles/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Colesterol/farmacología , Estrona/análogos & derivados , Estrona/metabolismo , Glicosilación , Humanos , Cinética , Metotrexato/metabolismo , Proteínas de Neoplasias/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Preparaciones Farmacéuticas/metabolismo , Prazosina/metabolismo , Spodoptera , Sulfasalazina/metabolismo , Topotecan/metabolismoRESUMEN
The importance of P-glycoprotein (P-gp) in the pharmacokinetics of amisulpride and the effects of a P-gp inhibitor cyclosporine A (CsA) was investigated both, in vitro and in vivo. In vitro and in vivo results indicated amisulpride as a substrate of P-gp. Amisulpride was not metabolized by rat liver microsomes. Open field behavior showed time dependent abolishment in locomotion by amisulpride (50 mg kg(-1)). Co-administration of CsA (50 mg kg(-1)) resulted in a higher and significantly longer antipsychotic effect (24 h after drug administration). Accordingly, the area under concentration-time curve in serum and brain was higher in CsA co-treated rats (13.5 vs. 29.8 micromol h l(-1) for serum and 2.16 vs 2.98 micromol h l(-1) for brain tissue) while renal clearance was not affected. These results pointed to a pharmacokinetic drug interaction between CsA and amisulpride most likely caused by inhibition of P-gp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Ciclosporina/farmacocinética , Sulpirida/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Amisulprida , Animales , Antipsicóticos/sangre , Antipsicóticos/farmacocinética , Antipsicóticos/orina , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Células CACO-2 , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas/fisiología , Inhibidores Enzimáticos/farmacocinética , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Ensayo de Unión Radioligante , Ratas , Sulpirida/sangre , Sulpirida/farmacocinética , Sulpirida/orina , Factores de TiempoRESUMEN
Chemical genomics, which utilizes specially designed small chemical compounds early in the discovery phase of new drugs to explore the life science at various levels, can address biological questions that are not amenable to genetic manipulation or functional genomics/proteomics approaches. Following the development of HT phenotypic assays and DNA expression analysis, the integration of cell-based assays with activity / affinity-based approaches allows us to interrogate the cells by analyzing phenotypic alterations, changes of transcript signature or detecting the differences in protein expression levels. Furthermore, activity / affinity-based techniques directly provide a druggable subset of gene products, which interact with small molecules, greatly reducing the complexity of analyzing the proteome. In this paper, we give an account of the recent advances (approaches and strategies) in the field of chemical genomics, and discuss how these approaches enable the investigator to obtain a novel therapeutically relevant target as well as drug candidates acting on them in a target-specific manner. This novel post-genomic discovery strategy, where target identification/ validation is carried out by interactions with small molecules, could significantly reduce the time-scale for early drug discovery, and increase the success rate of finding novel, druggable targets, as well as more specific drug candidates.
Asunto(s)
Ingeniería Química/métodos , Ingeniería Química/tendencias , Genómica/métodos , Genómica/tendencias , Animales , Regulación de la Expresión Génica/fisiología , Humanos , Ligandos , Unión Proteica/fisiologíaRESUMEN
We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.
Asunto(s)
Adenovirus Humanos/genética , Terapia Genética , Vectores Genéticos , Neoplasias/terapia , Replicación Viral , Proteínas E1A de Adenovirus/genética , Proteínas E4 de Adenovirus/biosíntesis , Adenovirus Humanos/fisiología , Animales , Replicación del ADN , Humanos , Ratones , Especificidad de Órganos , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
Cell death is common to many pathological conditions. In the past two decades, research into the mechanism of cell death has characterized the cardinal features of apoptosis and necrosis, the two distinct forms of cell death. Studies using in vivo disease models have provided evidence that apoptosis is induced by an array of pathological stimuli. Thus, molecular components of the machinery of apoptosis are potential pharmacological targets. The mechanism of apoptosis can be dissected into: (i) the initiation and signaling phase, (ii) the signal amplification phase, and (iii) the execution phase. Reflecting on the diversity of apoptotic stimuli, the initiation and signaling phase utilizes a variety of molecules: free radicals, ions, plasma membrane receptors, members of the signaling kinase cascades, transcription factors, and signaling caspases. In most of the apoptotic scenarios, impairment of mitochondrial function is an early event. Dysfunctioning mitochondria release more free radicals and hydrolytic enzymes (proteases and nucleases), amplifying the primary death signal. In the final phase of apoptosis, executioner caspases are activated. Substrates of the executioner caspases include nucleases, members of the cellular repair apparatus, and cytoskeletal proteins. Partial proteolysis of these substrates leads to distinctive morphological and biochemical changes, the hallmarks of apoptosis. The first steps toward pharmacological utilization of specific modifiers of apoptosis have been promising. However, since the potential molecular targets of cytoprotective therapy play important roles in the maintenance of cellular homeostasis, specificity (diseased versus healthy tissue) of pharmacological modulation is the key to success.
Asunto(s)
Citoprotección/fisiología , Animales , Apoptosis/fisiología , Caspasas/fisiología , Radicales Libres , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , FN-kappa B/fisiología , Óxido Nítrico Sintasa/fisiología , Poli(ADP-Ribosa) Polimerasas/fisiologíaRESUMEN
The effect of 3-nitrosobenzamide (NOBA) on the etoposide, staurosporine and dexamethason induced rapid (4-6 hr), caspase-dependent apoptosis was investigated in thymocytes and lymphoma cells by flow cytometric assay of DNA fragmentation. When NOBA (ED(50) = 4 microM) was added to these cell systems, the rapid onset of apoptosis was prevented. Such apparent protection by NOBA was related to the inactivation of caspase-3, by s-nitrosylation of 1.3 mol -SH per enzyme molecule out of 7 -SH groups. Since NOBA by itself induces DNA fragmentation within 18 hr in lymphoma cells, our results indicate that at least two active cell death pathways exist with apparent dissimilar kinetics and molecular mechanisms.
Asunto(s)
Benzamidas/farmacología , Inhibidores de Caspasas , Dexametasona/toxicidad , Etopósido/toxicidad , Compuestos Nitrosos/farmacología , Linfocitos T/inmunología , Animales , Caspasa 3 , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fragmentación del ADN , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/antagonistas & inhibidores , Citometría de Flujo , Humanos , Cinética , Linfoma de Células B , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
DNA viruses use elegant mechanisms to overcome the antiviral responses mediated by tumor necrosis factor (TNF), the TNF receptor family member Fas and the interferons. TNF, which is secreted by activated monocytes and lymphocytes, induces apoptosis as well as expression of genes involved in the inflammatory and immune responses. Depending on the DNA virus and the viral proteins, the following mechanisms to prevent TNF receptor- and Fas-induced apoptosis are used: (1) absorption of extracellular TNF by secreted homologs of the TNF receptor; (2) degradation of Fas; (3) inhibition of the assembly of FADD and Caspase 8 with TNFR1 and Fas; (4) direct inhibition of proapoptotic caspase enzymatic activity; and (5) inhibition of the proapoptotic members of the Bcl-2 family. Interferons induce expression of multiple antiviral genes. DNA viruses encode proteins that function in different ways to block interferon-induced transcription as well as the activity of enzymes that block viral protein synthesis. These antiviral proteins prolong acute and persistent infections.
Asunto(s)
Apoptosis/inmunología , Virus ADN/inmunología , Interferones/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Proteína Ligando Fas , Ligandos , Glicoproteínas de Membrana/inmunología , Proteínas Virales/inmunologíaAsunto(s)
Transducción de Señal , Proteínas Virales/fisiología , Animales , Muerte Celular , División Celular , HumanosRESUMEN
We have reported that three adenovirus (Ad) proteins, named E3-10.4K/14.5K, E3-14.7K, and E1B-19K, independently inhibit tumor necrosis factor (TNF)-induced apoptosis in Ad-infected cells. E3-10.4K/14.5K and E3-14.7K also inhibit TNF-induced release of arachidonic acid (AA). TNF-induced apoptosis and AA release are thought to require TNF-activation of the 85-kDa cytosolic phospholipase A2 (cPLA2). cPLA2 normally exists in a latent form in the cytosol; it is activated by phosphorylation by mitogen-activated protein kinase, and in the presence of agents that mobilize intracellular Ca2+, cPLA2 translocates to membranes where it cleaves AA from membrane phospholipids. We now report that TNF induces translocation of cPLA2 from the cytosol to membranes in Ad-infected human A549 cells and that E3-10.4K/14.5K but not E3-14.7K or E1B-19K is required to inhibit TNF-induced translocation of cPLA2. Ad infection also inhibited TNF-induced release of AA. Under the same conditions, Ad infection did not inhibit TNF-induced phosphorylation of cPLA2 or TNF activation of NFkappaB. Ad infection also inhibited cPLA2 translocation in response to the Ca2+ ionophore A23187 and to cycloheximide, but this inhibition did not require E3-10.4K/14.5K. Ad infection did not inhibit cPLA2 translocation in response to interleukin-1beta or platelet-derived growth factor. We propose that E3-10.4K/14.5K inhibits TNF-induced AA release and apoptosis by directly or indirectly inhibiting TNF-induced translocation of cPLA2 from the cytosol to membranes. AA formed by cPLA2 can be metabolized to prostaglandins, leukotrienes, and lipoxyns, molecules that amplify inflammation. E3-10.4K/14.5K probably functions in Ad infections to inhibit both TNF-induced apoptosis and inflammation.
Asunto(s)
Proteínas E3 de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Fosfolipasas A/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis , Transporte Biológico , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Fosfolipasas A2 , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Energy charge controls intermediary metabolism and cellular regulation. Here we show that inhibition of energy conservation at the level of glucose uptake, glycolysis, citric acid cycle, and oxidative phosphorylation induces cell death, leading to fragmentation of DNA into an oligonucleosomal ladder and morphological changes typical for apoptosis. Bcl-2, the prototype of oncogenes that suppress cell death, efficiently inhibits apoptosis induced by metabolic inhibitors. Bcl-2 does not antagonize the inhibitory potential of mitochondrial inhibitors, and cannot prevent or delay the decrease of the cellular ATP level subsequent to metabolic inhibition. Thus, we propose that Bcl-2 blocks apoptosis at a point downstream of the collapse of the cellular-energy homeostasis.
Asunto(s)
Adenosina Trifosfato/análisis , Apoptosis/efectos de los fármacos , Metabolismo Energético , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Línea Celular , Transporte de Electrón , Glucólisis , Ratones , Mitocondrias/metabolismoRESUMEN
Tumor necrosis factor (TNF) is an inflammatory cytokine that inhibits the replication of many viruses in cultured cells. We have reported that adenovirus (Ad) infection of TNF-resistant mouse cells renders them susceptible to lysis by TNF and that two sets of proteins encoded by the E3 transcription unit block TNF cytolysis. The E3 protein sets are named E3-14.7K (14,700 kDa) and E3-10.4K/14.5K (a complex of two proteins of 10,400 and 14,500 kDa). TNF activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) is thought to be essential for TNF cytolysis (i.e.,TNF-induced apoptosis). Here we provide evidence that cPLA2 is important in the response of Ad-infected cells to TNF and that the mechanism by which E3-14.7K and E3-10.4K/14.5K inhibit TNF cytolysis is by inhibiting TNF activation of cPLA2. cPLA2 cleaves arachidonic acid (AA) specifically from membrane phospholipids; therefore, cPLA2 activity was measured by the release of 3H-AA from cells prelabeled with 3H-AA. Uninfected cells or cells infected with wild-type Ad were not lysed and did not release 3H-AA in response to TNF. In contrast, TNF treatment induced cytolysis and 3H-AA release in uninfected cells sensitized to TNF by treatment with cycloheximide and also in infected cells sensitized to TNF by expression of E1A. In C127 cells, in which either E3-14.7K or E3-10.4K/14.5K inhibits TNF cytolysis, either set of proteins inhibited TNF-induced release of 3H-AA. In C3HA cells, in which E3-14.7K but not E3-10.4K/14.5K prevents TNF cytolysis, E3-14.7K but not E3-10.4K/14.5K prevented TNF-induced release of 3H-AA. When five virus mutants with lesions in E3-14.7K were examined, there was a perfect correlation between a mutant's ability to inhibit both TNF-induced cytolysis and release of 3H-AA. E3-14.7K expressed in two stably transfected C127 cell lines prevented both TNF-cycloheximide-induced cytolysis and release of 3H-AA. The E3 proteins also prevented TNF-induced cytolysis and release of 3H-AA in mouse L929 cells, which are spontaneously sensitive to TNF. TNF cytolysis was blocked by dexamethasone, an inhibitor of PLA2 activity, and by nordihydroquaiaretic acid, which inhibits the metabolism of AA to the leukotrienes. Indomethacin, which blocks the formation of prostaglandins from AA, did not inhibit TNF cytolysis. The leukotrienes and prostaglandins are amplifiers of the inflammatory response. We propose that E3-14.7K and E3-10.4K/14.5K function independently in Ad infection to inhibit both cytolysis and inflammation induced by TNF.
Asunto(s)
Infecciones por Adenoviridae , Proteínas E3 de Adenovirus/farmacología , Apoptosis/efectos de los fármacos , Ácido Araquidónico/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Infecciones por Adenoviridae/metabolismo , Infecciones por Adenoviridae/patología , Animales , Línea Celular , Ratones , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor.
Asunto(s)
Proteínas E3 de Adenovirus/fisiología , Receptores ErbB/metabolismo , Proteínas de la Membrana/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Secuencia de Aminoácidos , Muerte Celular , Células Cultivadas , Regulación hacia Abajo , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Fracciones Subcelulares/metabolismoRESUMEN
The adenovirus E3-14.5K protein is a cytoplasmic integral membrane protein that functions in concert with the E3-10.4K protein to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. The 14.5K protein migrates as multiple bands in SDS-PAGE, indicating that it undergoes post-translational modification. The 14.5K protein is known to be phosphorylated on serine. We show here that 14.5K can be metabolically labeled with [3H]glucosamine, that the label is labile to alkali, and that the SDS-PAGE band pattern is simplified in a cell line that is defective in O-glycosylation. Thus, 14.5K is O-glycosylated, probably at a single site in the NH2-terminal lumenal domain. The protein was not metabolically labeled with [3H]mannose, and its SDS-PAGE band pattern was not affected by tunicamycin treatment in vivo or endo F treatment in vitro; thus, 14.5K is not N-glycosylated. There was no evidence that the 10.4K protein is glycosylated, and the 10.4K protein was not required for glycosylation of 14.5K. Virtually all 14.5K molecules appear to contain the core disaccharide Gal beta 1-3GalNAc alpha 1-Ser/Thr which is commonly found on mucin-type O-glycoproteins, and neuraminidase digestion experiments indicated that this disaccharide contains terminal sialic acid.
Asunto(s)
Adenovirus Humanos/química , Glicoproteínas de Membrana/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Precoces de Adenovirus , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Células Cultivadas , Regulación hacia Abajo , Receptores ErbB/metabolismo , Glicosilación , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
The E3-14.5K and E3-10.4K proteins form a complex and function to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. Both 14.5K and 10.4K are cytoplasmic membrane proteins with a Ccyt orientation in the membrane. We show here that 14.5K is phosphorylated on serine residues in cells infected by adenoviruses that synthesize both 14.5K and 10.4K. 14.5K is phosphorylated on both serine and threonine in cells infected by a mutant that does not synthesize 10.4K; thus, the presence or absence of 10.4K affects the phosphorylation of 14.5K. Phosphotyrosine was not detected. 14.5K is also phosphorylated when translated in vitro in a rabbit reticulocyte extract. Both in vivo and in vitro, at least one of the phosphorylation sites is near the C-terminus, in the cytoplasmic domain of 14.5K. This C-terminal region of 14.5K is the most conserved among Ad5, Ad2, Ad3, and Ad7, and it is essential for 14.5K to prevent tumor necrosis factor cytolysis.
Asunto(s)
Adenovirus Humanos/química , Receptores ErbB/metabolismo , Proteínas Oncogénicas Virales/química , Fosfoserina/química , Proteínas Precoces de Adenovirus , Adenovirus Humanos/metabolismo , Secuencia de Aminoácidos , Regulación hacia Abajo , Humanos , Técnicas In Vitro , Células KB , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/metabolismo , Mapeo Peptídico , Fosforilación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The Ad2 E3-10.4K protein is required together with the E3-14.5K protein to down-regulate the epidermal growth factor receptor in adenovirus-infected cells. Both proteins are also required to prevent tumor necrosis factor cytolysis under certain conditions. 10.4K is a 91 amino acid membrane-associated protein that migrates as two bands, upper and lower, on SDS-PAGE. We show here that the upper band is the primary translation product which initiates at AUG2173 in the E3 transcription unit of Ad2. The upper band is processed slowly (greater than 4 hr to complete) into the lower band by proteolytic cleavage between residues Ala22 and Ala23 by a microsome-associated protease. The upper and lower bands become equal in abundance, after which they are very stable. The N-terminus of the in vivo-derived upper band is not blocked to sequencing and it retains its initiating Met. 10.4K has a hydrophobic domain (H1) near its N-terminus that is probably a signal sequence for membrane insertion; cleavage of this signal is atypical because it was not cotranslational in vivo and it was not complete. 10.4K has a second hydrophobic domain (H2) located within residues 35-60. H2 appears to be a transmembrane (stop transfer) domain because both the upper and the lower 10.4K bands remained associated with membranes after extraction at pH 11.5, because both bands were extracted into the detergent phase with Triton X-114, and because both bands were only partially reduced in size when 10.4K-containing microsomes were digested with proteinase K. These proteinase K-digested bands were immunoprecipitated with an antipeptide antiserum against residues 19-34 but not with an antiserum against residues 68-80 or 77-91, indicating that both 10.4K bands are orientated in the membrane with the C-terminus in the cytoplasm. We conclude that the lower band of 10.4K is a type I bitopic membrane protein and suggest that the upper band is a polytopic membrane protein with both the H1 and the H2 hydrophobic domains spanning the membrane.
Asunto(s)
Adenovirus Humanos/metabolismo , Antígenos Virales de Tumores/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Precoces de Adenovirus , Adenovirus Humanos/química , Adenovirus Humanos/genética , Secuencia de Aminoácidos , Antígenos Virales de Tumores/química , Antígenos Virales de Tumores/genética , Línea Celular , Endopeptidasa K , Humanos , Microsomas/metabolismo , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Pruebas de Precipitina , Serina Endopeptidasas/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.