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1.
J Biol Chem ; 286(9): 7060-9, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21189259

RESUMO

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.


Assuntos
Estenose Coronária/metabolismo , Variação Genética , Receptores de Prostaglandina , Transdução de Sinais/fisiologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Sequência Conservada , Estenose Coronária/epidemiologia , Estenose Coronária/fisiopatologia , Feminino , Humanos , Iloprosta/farmacologia , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Receptores de Epoprostenol , Receptores de Prostaglandina/química , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Vasodilatadores/farmacologia , Adulto Jovem
2.
Circ Res ; 102(8): 986-93, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18323528

RESUMO

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition.


Assuntos
Doenças Cardiovasculares/genética , Inibidores de Ciclo-Oxigenase 2/efeitos adversos , Mutação de Sentido Incorreto , Receptores de Epoprostenol/genética , Doenças Cardiovasculares/patologia , Estudos de Casos e Controles , Progressão da Doença , Humanos , Receptores Acoplados a Proteínas G , Transdução de Sinais
3.
J Mol Cell Cardiol ; 46(5): 682-94, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19302827

RESUMO

The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Iloprosta/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Aorta/citologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Indução Enzimática/efeitos dos fármacos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos de Músculo Liso/enzimologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Epoprostenol , Receptores de Prostaglandina/metabolismo , Regulação para Cima/efeitos dos fármacos
4.
Gene ; 396(1): 180-7, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17481829

RESUMO

The human prostacyclin receptor (hIP) has recently been recognized as an important seven transmembrane G-protein coupled receptor that plays critical roles in atheroprevention and cardioprotection. To date, four non-synonymous genetic variants have been identified, two of which occur at the same Arg amino acid position (R212H, R212C). This observation instigated further genetic screening for prostacyclin receptor variants on 1455 human genomic samples. A total of 31 distinct genetic variants were detected, with 6 (19%) involving Arg residues. Distinct differences in location and frequencies of genetic variants were noted between Caucasian, Asian, Hispanic and African Americans, with the most changes noted in the Asian cohort. From the sequencing results, three Arg-targeted changes at the same 212 position within the third cytoplasmic loop of the human prostacyclin (hIP) receptor were detected: 1) R212C (CGC-->TGC), 2) R212H (CGC-->CAC), and 3) R212R (CGC-->CGT). Three additional Arg codon variants (all exhibiting the same CGC to TGC change) were also detected, R77C, R215C, and R279C. Analysis (GPCR and SNP databases) of 200 other GPCRs, with recorded non-synonymous mutations, confirmed a high frequency of Arg-targeted missense mutations, particularly within the important cytoplasmic domain. Preferential nucleotide changes (at Arg codons), were observed involving cytosine (C) to thymine (T) (pyrimidine to pyrimidine), as well as guanine (G) to adenine (A) (purine to purine) (p<0.001, Pearson's goodness-of-fit test). Such targeting of Arg residues, leading to significant changes in coding amino acid size and/or charge, may have potentially-important structural and evolutionary implications on the hIP and GPCRs in general. In the case of the human prostacyclin receptor, such alterations may reduce the cardio-, vasculo-, and cytoprotective effects of prostacyclin.


Assuntos
Arginina/genética , Códon/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Prostaglandina/genética , Sequência de Aminoácidos , Sequência de Bases , Citoplasma/metabolismo , Bases de Dados Genéticas , Genoma Humano/genética , Humanos , Dados de Sequência Molecular , Nucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Epoprostenol , Receptores de Prostaglandina/química , Análise de Sequência de DNA
5.
Infect Dis Clin North Am ; 21(1): 219-39, x, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17502237

RESUMO

Topical microbicides are self-administered products for prevention of HIV transmission, and they present one of the most promising strategies for combating the HIV-AIDS epidemic. The development of microbicides is a long and complicated process, with many hurdles that are unique to this class of product, including challenges in product design, in the conduct and design of clinical trials, and in obtaining licensure of a new class of products intended for use almost exclusively in developing countries. Once they have been registered, there are additional challenges to the marketing and distribution of microbicides. An overview of the types of microbicide currently in development, and a summary of the issues and the approaches being taken to address them, are provided.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacologia , Anti-Infecciosos Locais/farmacologia , Infecções por HIV/prevenção & controle , Administração Tópica , Anti-Infecciosos Locais/administração & dosagem , Antagonistas dos Receptores CCR5 , Ensaios Clínicos como Assunto , Células Dendríticas/efeitos dos fármacos , Surtos de Doenças , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Humanos , Receptores CXCR4/antagonistas & inibidores , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/farmacologia
6.
Prostaglandins Other Lipid Mediat ; 82(1-4): 95-108, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17164137

RESUMO

Prostacyclin (PGI2) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation (anti-thrombotic), and moderator of vascular smooth muscle cell proliferation-migration-differentiation (anti-atherosclerotic). These actions are mediated via a seven transmembrane-spanning G-protein coupled receptor (GPCR), known as the human prostacyclin receptor or hIP. Animal studies using prostacyclin receptor knock-out (IP-/-) mice have revealed increased propensities towards thrombosis, intimal hyperplasia, atherosclerosis, restenosis, as well as reperfusion injury. Of further importance has been the world-wide withdrawal of selective COX-2 inhibitors, due to their discriminating suppression of COX-2-derived PGI2 and its cardioprotective effects, leading to increased cardiovascular events, including myocardial infarction and thrombotic stroke. Over the last decade, mutagenesis studies of the IP receptor, in conjunction with in vitro functional assays and molecular modeling, have provided critical insights into the molecular mechanisms of both agonist binding and receptor activation. Most recently, the discovery of naturally-occurring and dysfunctional mutations within the hIP has provided additional insights into the proposed cardioprotective role of prostacyclin. The aim of this review is to summarize the most recent findings regarding hIP receptor structure-function that have developed through the study of both synthetic and naturally-occurring mutations.


Assuntos
Receptores de Epoprostenol/química , Receptores de Epoprostenol/fisiologia , Sequência de Aminoácidos , Asparagina/química , Sítios de Ligação , Cisteína/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ácido Palmítico/metabolismo , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Prolina/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Epoprostenol/genética , Serina/química
7.
Oncogene ; 21(1): 108-18, 2002 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-11791181

RESUMO

To characterize the impact of increased production of TGF-beta in a xenograft model of human breast cancer, TGF-beta-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-beta1. Compared with control cells, cells that produced increased amounts of TGF-beta proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-beta signaling, cells were also transfected with a dominant-negative truncated type II TGF-beta receptor (TbetaRII). Disruption of autocrine TGF-beta signaling in the TGF-beta-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-beta-overexpressing cells expressing the truncated TbetaRII compensated for their diminished in vivo growth capacity, compared with the TGF-beta-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-beta-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-beta-overexpressing cells, irrespective of the status of the autocrine TGF-beta-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-beta's enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-beta that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.


Assuntos
Comunicação Autócrina , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Comunicação Parácrina , Fator de Crescimento Transformador beta/fisiologia , Animais , Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/complicações , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/secundário , Divisão Celular , Colágeno , Meios de Cultivo Condicionados/farmacologia , Combinação de Medicamentos , Feminino , Genes Dominantes , Hemorragia/etiologia , Humanos , Laminina , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases , Proteoglicanas , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Úlcera Cutânea/etiologia , Transfecção , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Transplante Heterólogo , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia
8.
AIDS Res Hum Retroviruses ; 21(10): 845-53, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16225411

RESUMO

The development of vaginal microbicides for the prevention of sexual transmission of HIV is becoming an increasingly important strategy in the battle against the AIDS epidemic. Several first generation microbicide candidates are entering Phase III efficacy trials, and several other candidates are in earlier stages of clinical development. The capacity to make accurate clinical assessments of the safety and efficacy of microbicide formulations is critical. Since microbicide trials will rely on a blinded, randomized, placebo-controlled design, it is important to employ a placebo formulation that does not distort either safety or efficacy assessments. Efficacy of the microbicide would be underestimated if the placebo itself provided a degree of protection. Conversely, a placebo with epithelial toxicity that increased susceptibility would cause an overestimation of microbicide efficacy. To address these issues, a hydroxyethylcellulose (HEC) placebo formulation has been developed and has been adopted for use in clinical evaluations of investigational microbicides as a "universal" placebo. In this report, the chemical and physical properties of this formulation are described, as well as its in vitro and in vivo effects on safety and efficacy. The results show that this "universal" placebo has adequate physical properties, is sufficiently stable as a vaginal gel formulation, and is safe and sufficiently inactive for use in the clinical study of investigational microbicides.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Anti-Infecciosos Locais/administração & dosagem , Infecções por HIV/prevenção & controle , Placebos , Ensaios Clínicos Controlados Aleatórios como Assunto , Vagina , Animais , Feminino , Infecções por HIV/transmissão , Humanos , Concentração de Íons de Hidrogênio , Macaca , Coelhos , Viscosidade
9.
PLoS One ; 8(6): e67314, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840660

RESUMO

Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C(68)S, V(80)E, E(94)V, A(160)T, V(176)E, and V(217)I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C(68)S) to normal (V(217)I), with most variants demonstrating functional alteration in binding, expression or activation (V(80)E, E(94)V, A(160)T, and V(176)E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and "in platelet" evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.


Assuntos
Plaquetas/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aspirina/farmacologia , Sítios de Ligação , Ligação Competitiva , Plaquetas/efeitos dos fármacos , Linhagem Celular , Inibidores de Ciclo-Oxigenase/farmacologia , Estudos de Associação Genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteoma/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/química , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Transdução de Sinais , Tromboxanos/fisiologia
10.
Sex Transm Dis ; 35(4): 414-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18362865

RESUMO

GOAL: This study evaluated the effect of a single dose and 5 additional consecutive daily doses of UC781 gel at concentrations of 0.1%, 0.25%, 1.0%, and 0% on urogenital irritation. STUDY DESIGN: Forty-eight healthy sexually abstinent women were randomly assigned to 1 of 4 groups. METHODS: Urogenital irritation was assessed by pelvic examination, colposcopy, and reports of genital symptoms at baseline and after 1 and 6 doses. Vaginal health was assessed by wet mount and systemic safety by laboratory evaluation after 1 and 6 doses, and UC781 levels were assessed at baseline and after 6 doses. RESULTS: Some evidence of urogenital irritation was common in all treatment groups and was most often transient and mild. Colposcopic findings were infrequent in the placebo group (8%) and more common in the 3 treatment groups (24%-42%). Edema, which may indicate underlying inflammation, was observed in the vaginal fornix of 2 women exposed to UC781. There was no apparent increase in vaginal infection or clinically significant changes in laboratory values. Two of 12 participants randomized to 1% UC781 gel had detectable plasma levels that were less than the lower level of quantification. CONCLUSIONS: UC781 was well tolerated in this initial dose ranging safety study when used once daily for 6 days in sexually abstinent women. Five safety/pharmacokinetic studies of UC781 are currently underway in women and men, all utilizing UC781 concentrations less than 1%, with twice-daily dosing in some studies, and all involving careful monitoring of exposed epithelium.


Assuntos
Anilidas/administração & dosagem , Furanos/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Sistema Urogenital/efeitos dos fármacos , Administração Intravaginal , Adulto , Anilidas/efeitos adversos , Colposcopia , Feminino , Furanos/efeitos adversos , Infecções por HIV , Humanos , Pessoa de Meia-Idade , Satisfação do Paciente , Inibidores da Transcriptase Reversa/efeitos adversos , Tioamidas , Resultado do Tratamento , Cremes, Espumas e Géis Vaginais/administração & dosagem
11.
Pharmacogenet Genomics ; 18(7): 611-20, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18551041

RESUMO

OBJECTIVE AND METHODS: The role of prostacyclin in the development of venous thrombosis and vascular dysfunction in humans is unclear. In patients with deep vein thrombosis (DVT, n=34) and controls (matched for age, sex, indexes of systemic inflammation and metabolic status, n=20), we studied (i) differences on systemic markers of vascular disease and platelet activation and (ii) the influence of prostacyclin receptor gene (PTGIR) polymorphisms. MAIN RESULTS: Enhanced levels of urinary 11-dehydro-thromboxane (TX)B2 and plasma [soluble(s)] P-selectin, mostly platelet derived, were detected in DVT patients, whereas plasma von Willebrand factor levels and intima-media thickness of the common carotid arteries were not significantly different. In all patients' cohorts, we identified five PTGIR polymorphisms (three nonsynonymous: P226T, R212C, V196L; two synonymous: V53V, S328S). In the four individuals carriers of R212C polymorphism (three in DVT, one in controls), intima-media thickness values were significantly (P=0.0043) higher than those detected in individuals of all cohorts [1.68+/-0.38, 1.55 (1.4-2.2) vs. 1.05+/-0.33, 1.08 (0.01-1.68) mm, respectively, mean+/-SD, median (range)]. Moreover, enhanced sP-selectin and 11-dehydro-TXB2, in DVT versus controls, were statistically significant only in carriers of both synonymous PTGIR polymorphisms V53V/S328S. Only the PTGIR mutant R212C was dysfunctional when examined in an in vitro overexpression system. CONCLUSION: Our results suggest a propensity of enhanced platelet activation in DVT patients with PTGIR polymorphisms V53V/S328S. Moreover, we identified a dysfunctional PTGIR polymorphism (R212C) associated with intimal hyperplasia.


Assuntos
Biomarcadores/análise , Polimorfismo de Nucleotídeo Único , Receptores de Epoprostenol/genética , Túnica Íntima/patologia , Trombose Venosa/genética , Adulto , Idoso , Feminino , Ligação Genética , Testes Genéticos , Humanos , Hiperplasia/genética , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Ativação Plaquetária/genética , Tromboxano B2/análogos & derivados , Tromboxano B2/urina , Trombose Venosa/sangue , Trombose Venosa/patologia , Trombose Venosa/urina
12.
J Biol Chem ; 281(48): 37227-36, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17015447

RESUMO

Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).


Assuntos
Cisteína/química , Receptores de Epoprostenol/química , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
13.
J Cell Biochem ; 89(1): 120-32, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12682913

RESUMO

The global changes in gene expression induced by transient increased expression of full length BRCA1 as well as the spliced variant BRCA1(S) were evaluated by cDNA expression array in a human non-tumorigenic mammary epithelial cell line, MCF10A. Over 30 genes were identified that displayed an altered expression pattern in response to the expression of BRCA1 splice variants. The expression of NFkappaB inducing kinase was markedly down-regulated in BRCA1(L) transfected cells. However, a NFkappaB-responsive promoter construct yielded increased basal activity in BRCA1(L) transfected cells, as well as following treatment with tumor necrosis factor-alpha or lymphotoxin. In addition, nuclear extracts from BRCA1(L) transfected cells displayed increased DNA binding to the kappaB consensus site. The transcriptional activity of a panel of promoter constructs was evaluated following expression of wild type or mutant BRCA1. Full length BRCA1 transactivated the estrogen receptor-alpha (ERalpha) and BCL2 promoters as well as AP-1, SRE, and CRE containing promoters. Transactivation activity of the exon 11-deleted BRCA1(S) was more limited and usually of lower magnitude. The ability of a pathogenic mutation, 5382insC, to abrogate the transcriptional transactivation by BRCA1(L) and BRCA1(S) was also investigated. Mutant BRCA1 retained wild type levels of transcriptional activity for the ERalpha promoter as well as for the NFkappaB, AP-1, and CRE-responsive promoters but had reduced or no activity with the BCL2 and SRE promoters. These results show that BRCA1 isoforms have both overlapping and distinct transcriptional transactivation activity, and that a mutant form of BRCA1 implicated in carcinogenesis is not devoid of all activity.


Assuntos
Processamento Alternativo , Genes BRCA1 , Sequência de Bases , Mama/citologia , Mama/metabolismo , Linhagem Celular , DNA Complementar/genética , Feminino , Expressão Gênica , Humanos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ativação Transcricional , Transfecção , Quinase Induzida por NF-kappaB
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