RESUMO
Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.
Assuntos
Apolipoproteína A-I/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Animais , Apolipoproteína A-I/genética , HDL-Colesterol/genética , HDL-Colesterol/metabolismo , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/genética , Farnesiltranstransferase/metabolismo , Estudo de Associação Genômica Ampla , Células Hep G2 , Humanos , Fígado/citologia , Camundongos , Camundongos Transgênicos , Polienos/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , RNA Interferente Pequeno/genéticaRESUMO
The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.
Assuntos
Aciltransferases/metabolismo , Grelina/metabolismo , Acilação , Aciltransferases/genética , Animais , Caprilatos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Sequência Conservada , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Grelina/sangue , Grelina/genética , Humanos , Dados de Sequência Molecular , Pâncreas/enzimologia , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/metabolismo , Estômago/enzimologiaRESUMO
Elimination of an enzootic infection of mouse hepatitis virus (MHV) from a large population of genetically engineered mice was accomplished by selecting seropositive, non-infective breeders for a newly restored MHV-free breeding colony. An ELISA was used to test for the presence of MHV-specific antibody, and TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to detect MHV in the feces. After 10 weeks of intentional exposure, approximately 30% of mice with MHV antibodies continued to shed MHV in the feces. A natural transmission study was conducted to validate that positive fecal RT-PCR results indicated presence of infective virus. Sentinel results from the re-instituted breeding colony indicated that MHV was successfully eliminated by use of RT-PCR analysis for selection of non-infective mice.
Assuntos
Surtos de Doenças/veterinária , Hepatite Viral Animal/diagnóstico , Vírus da Hepatite Murina/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Taq Polimerase/metabolismo , Animais , Anticorpos Antivirais/imunologia , Cruzamento , Surtos de Doenças/prevenção & controle , Ensaio de Imunoadsorção Enzimática/veterinária , Fezes/virologia , Feminino , Hepatite Viral Animal/prevenção & controle , Hepatite Viral Animal/transmissão , Masculino , Camundongos , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/imunologia , Vírus da Hepatite Murina/isolamento & purificação , RNA Mensageiro/análise , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Taq Polimerase/genéticaRESUMO
The hormone ghrelin is a unique signaling peptide with powerful metabolic effects, mediated by its acylated forms. The acyl modification of ghrelin is unique in that it takes place via a susceptible ester linkage in the conserved serine-3 of ghrelin and is composed principally of octanoyl and, to lesser extent, decanoyl fatty acids. The nature of this ester linkage makes it susceptible to esterases, which convert it to its des-acyl forms, and, if not adequately inhibited, the conversion to des-acyl ghrelin, particularly post sample collection, can lead to artifactual and misleading results. Here, we describe sample processing and mass spectrometric methodologies for the accurate and simultaneous quantification of acylated and des-acylated forms of ghrelin. We exploited these methodologies (1) to characterize circulating and tissue-specific forms of acyl and des-acyl ghrelin, (2) to optimize a cell system for acyl ghrelin production and search for the enzyme responsible for ghrelin's acylation, and (3) to demonstrate that GOAT is ghrelin's O-acyl transferase.
Assuntos
Aciltransferases/metabolismo , Técnicas de Cultura de Células/métodos , Grelina/sangue , Acilação , Aciltransferases/genética , Animais , Caprilatos/metabolismo , Linhagem Celular Tumoral , Meios de Cultura/metabolismo , Mucosa Gástrica/metabolismo , Inativação Gênica , Grelina/genética , Grelina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Estabilidade Proteica , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estômago/citologia , TransfecçãoRESUMO
Central nervous system nutrient sensing and afferent endocrine signaling have been established as parallel systems communicating metabolic status and energy availability in vertebrates. The only afferent endocrine signal known to require modification with a fatty acid side chain is the orexigenic hormone ghrelin. We find that the ghrelin O-acyl transferase (GOAT), which is essential for ghrelin acylation, is regulated by nutrient availability, depends on specific dietary lipids as acylation substrates and links ingested lipids to energy expenditure and body fat mass. These data implicate the ghrelin-GOAT system as a signaling pathway that alerts the central nervous system to the presence of dietary calories, rather than to their absence as is commonly accepted.
Assuntos
Aciltransferases/fisiologia , Gorduras na Dieta/administração & dosagem , Metabolismo Energético , Grelina/fisiologia , Transdução de Sinais/fisiologia , Aciltransferases/genética , Animais , Grelina/sangue , Grelina/genética , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/análise , Triglicerídeos/uso terapêuticoRESUMO
Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.
Assuntos
Artrite Experimental/sangue , Artrite Reumatoide/sangue , Biomarcadores/sangue , Perfilação da Expressão Gênica , Monócitos/metabolismo , Animais , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Feminino , Galectina 3/sangue , Galectina 3/metabolismo , Camundongos , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Glycinamide ribonucleotide formyltransferase (GARFT) is a trifunctional enzyme involved in purine biosynthesis. Its central role in folate metabolism has made it an obvious target for the development of GARFT inhibitors, primarily for oncology. While the crystal structure, enzyme kinetics, and mechanism of action of GARFT inhibitors are reasonably well understood, GARFT regulation at the protein level remains unclear. The present study reports the development and characterization of a monoclonal antibody (MAb) specific for human GARFT. This MAb, an IgG1kappa, designated PHR1, recognizes human GARFT by both Western blot and by immunohistochemistry from non-small-cell lung carcinoma and colon adenocarcinoma tissue biopsies, has a KD of 1.14 x 10(10) M, and has been epitope mapped at residues 59-78 of the GARFT functional domain. The ability of PHR1 to recognize both sodium dodecyl sulfate (SDS)-denatured as well as native GARFT should make this MAb an important research tool in determining GARFT protein levels in both normal and neoplastic tissues.
Assuntos
Anticorpos Monoclonais , Fosforribosilglicinamido Formiltransferase/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Epitopos/genética , Epitopos/imunologia , Células HeLa , Humanos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fosforribosilglicinamido Formiltransferase/genéticaRESUMO
BACKGROUND: Members of the nuclear receptor family proteins function as transcription factors upon ligand binding and thereby regulate gene expression in host cells. Aldosterone, the high-affinity endogenous ligand for the mineralocorticoid receptor, induces cardiac hypertrophy and fibrosis in a variety of animal models, but the transcriptional targets for aldosterone in the myocardium are not well-described. METHODS AND RESULTS: Using quantitative reverse transcription-polymerase chain reaction method, we show that in cultured rat neonatal cardiomyocytes, aldosterone stimulates expression of angiotensin converting enzyme (ACE) in a concentration and time-dependent manner. Aldosterone (50 and 100 nM) increased levels of ACE mRNA by 1.8- and 2.2-fold, respectively. Aldosterone-induced ACE gene expression was blocked by spironolactone (1 microM), a mineralocorticoid receptor antagonist. In contrast, the expressions of the type I angiotensin receptor was not induced by aldosterone in either cardiac myocytes or fibroblasts. Consistent with the increased ACE mRNA level, 100 nM aldosterone also induced a 2-fold increase in ACE activity in cardiac myocytes. CONCLUSION: ACE gene expression may be a target for mineralocorticoid receptors in the myocardium, supporting the notion that at least some of the known adverse effects of aldosterone on the myocardium are mediated by increased angiotensin II.