RESUMEN
A major goal in human genetics is to use natural variation to understand the phenotypic consequences of altering each protein-coding gene in the genome. Here we used exome sequencing1 to explore protein-altering variants and their consequences in 454,787 participants in the UK Biobank study2. We identified 12 million coding variants, including around 1 million loss-of-function and around 1.8 million deleterious missense variants. When these were tested for association with 3,994 health-related traits, we found 564 genes with trait associations at P ≤ 2.18 × 10-11. Rare variant associations were enriched in loci from genome-wide association studies (GWAS), but most (91%) were independent of common variant signals. We discovered several risk-increasing associations with traits related to liver disease, eye disease and cancer, among others, as well as risk-lowering associations for hypertension (SLC9A3R2), diabetes (MAP3K15, FAM234A) and asthma (SLC27A3). Six genes were associated with brain imaging phenotypes, including two involved in neural development (GBE1, PLD1). Of the signals available and powered for replication in an independent cohort, 81% were confirmed; furthermore, association signals were generally consistent across individuals of European, Asian and African ancestry. We illustrate the ability of exome sequencing to identify gene-trait associations, elucidate gene function and pinpoint effector genes that underlie GWAS signals at scale.
Asunto(s)
Bancos de Muestras Biológicas , Bases de Datos Genéticas , Secuenciación del Exoma , Exoma/genética , África/etnología , Asia/etnología , Asma/genética , Diabetes Mellitus/genética , Europa (Continente)/etnología , Oftalmopatías/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Hipertensión/genética , Hepatopatías/genética , Masculino , Mutación , Neoplasias/genética , Carácter Cuantitativo Heredable , Reino UnidoRESUMEN
While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Dineínas Axonemales/genética , HDL-Colesterol/sangre , Endorribonucleasas/genética , Mutación , Receptores Inmunológicos/genética , Transportador 1 de Casete de Unión a ATP/genética , Adulto , Anciano , Apolipoproteína A-I/genética , Proteínas de Transferencia de Ésteres de Colesterol/genética , HDL-Colesterol/genética , Femenino , Humanos , Lipasa/genética , Masculino , Persona de Mediana Edad , N-Acetilgalactosaminiltransferasas/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.
Asunto(s)
Modelos Animales de Enfermedad , Dislipidemias/sangre , Lípidos/sangre , Animales , Cricetinae , Perros , Dislipidemias/tratamiento farmacológico , Ácidos Grasos/sangre , Humanos , Ratones , Primates , Simvastatina/uso terapéutico , Triglicéridos/sangreRESUMEN
High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.
Asunto(s)
Ésteres del Colesterol/análisis , Colesterol/análisis , Cromatografía Liquida/métodos , Óxido de Deuterio/química , Espectrometría de Masas/métodos , Animales , Colesterol/sangre , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Isótopos , Masculino , Ratones , Ratones Endogámicos C57BL , Palmitatos/sangre , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoEâ»/â» and low density lipoprotein receptor (LDLr)â»/â» mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETPâº/â» hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETPâº/â» mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.
Asunto(s)
Apolipoproteínas B/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/genética , LDL-Colesterol/sangre , Modelos Animales de Enfermedad , Receptores de LDL/genética , Animales , Apolipoproteínas B/sangre , Apolipoproteínas E/sangre , Apolipoproteínas E/genética , Aterosclerosis/patología , Línea Celular Tumoral , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Efecto Fundador , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hemicigoto , Humanos , Metabolismo de los Lípidos/genética , Liposomas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanopartículas/administración & dosificación , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Receptores de LDL/metabolismo , Triglicéridos/sangreRESUMEN
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
Asunto(s)
LDL-Colesterol/sangre , Fragmentos Fab de Inmunoglobulinas/farmacología , Receptores de LDL/química , Serina Endopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Fluoroinmunoensayo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/metabolismo , Serina Endopeptidasas/químicaRESUMEN
The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of â¼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Cromatografía Liquida/métodos , Proteínas de Transporte de Ácidos Grasos/metabolismo , Hígado/patología , Espectrometría de Masas/métodos , Metabolómica/métodos , Animales , Silenciador del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The UK Biobank Exome Sequencing Consortium (UKB-ESC) is a private-public partnership between the UK Biobank (UKB) and eight biopharmaceutical companies that will complete the sequencing of exomes for all ~500,000 UKB participants. Here, we describe the early results from ~200,000 UKB participants and the features of this project that enabled its success. The biopharmaceutical industry has increasingly used human genetics to improve success in drug discovery. Recognizing the need for large-scale human genetics data, as well as the unique value of the data access and contribution terms of the UKB, the UKB-ESC was formed. As a result, exome data from 200,643 UKB enrollees are now available. These data include ~10 million exonic variants-a rich resource of rare coding variation that is particularly valuable for drug discovery. The UKB-ESC precompetitive collaboration has further strengthened academic and industry ties and has provided teams with an opportunity to interact with and learn from the wider research community.
Asunto(s)
Bancos de Muestras Biológicas , Descubrimiento de Drogas , Secuenciación del Exoma , Genética Humana , Investigación , Descubrimiento de Drogas/métodos , Genómica/métodos , Humanos , Reino UnidoRESUMEN
RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research.
Asunto(s)
Biotecnología/métodos , Genoma , Interferencia de ARN , Apolipoproteína A-I/genética , Biotecnología/normas , Hepacivirus/genética , Control de Calidad , Proyectos de Investigación/normasRESUMEN
Liver X receptor (LXR) alpha and LXRbeta are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRalpha and LXRbeta by synthetic compounds. We here describe the effects of selective activation of LXRalpha or LXRbeta on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRalpha or LXRbeta with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRalpha (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRalpha, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRalpha and LXRbeta-deficient mice. Our data lend further experimental support to the hypothesis that LXRbeta-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.
Asunto(s)
Proteínas de Unión al ADN/agonistas , Metabolismo de los Lípidos/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Colesterol/metabolismo , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , AMP Cíclico/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Isoxazoles/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Receptores X del Hígado , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Molecular , Receptores Nucleares Huérfanos , Compuestos de Fenilurea/farmacología , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Pirazinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery.
Asunto(s)
Prestación Integrada de Atención de Salud , Enfermedad/genética , Registros Electrónicos de Salud , Exoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Adulto , Diseño de Fármacos , Frecuencia de los Genes , Genómica , Humanos , Hipolipemiantes/farmacología , Mutación INDEL , Lípidos/sangre , Terapia Molecular Dirigida , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
Diabetes mellitus is one of the major risk factors associated with atherosclerosis and coronary heart disease but the mechanistic links between the disease and atherosclerosis are not well understood. In this study, we investigated the effect of the deletion of the long-form leptin receptor on the progression of atherosclerosis in ApoE-/- mouse. ApoE-/-;db/db double knockout mice as well as ApoE-/-;db/+ and ApoE-/- littermates were generated by crossing ApoE-/- and db/+ mice. On a regular chow diet, ApoE-/-;db/db mice at 20 weeks of age exhibited features typical of type 2 diabetes: obesity, hyperglycemia, hyperinsulinemia and dyslipidemia and had significantly accelerated atherosclerosis compared with their age-matched ApoE-/- littermates as assessed by either the percentage of the aorta bearing lesion (5.3+/-0.9% for ApoE-/-;db/db versus 1.5+/-0.5% for ApoE-/-) or by aortic lipid content ( approximately 1.5-2-fold increase in free cholesterol and approximately 3-4-fold increase in cholesteryl ester). The atherosclerosis in these ApoE-/-;db/db mice was further accelerated by feeding mice with a Western diet and markedly inhibited by fenofibrate with a 2.5- and 5.3-fold reduction of the lesion in male and female mice, respectively. The results from this study demonstrate that type 2 diabetes can accelerate atherogenesis in mice. This mouse model may provide insight into the mechanistic link between type 2 diabetes and atherosclerosis as well as serve as a valuable tool for evaluating therapeutics.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/genética , Modelos Animales de Enfermedad , Hipercolesterolemia/genética , Ratones Noqueados , Receptores de Superficie Celular/genética , Animales , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Colesterol/sangre , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/patología , Angiopatías Diabéticas/fisiopatología , Femenino , Fenofibrato/farmacología , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/fisiopatología , Hipolipemiantes/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Leptina , Índice de Severidad de la EnfermedadRESUMEN
Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.
RESUMEN
Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo. Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Hígado/metabolismo , Miniaturización/métodos , Receptores Citoplasmáticos y Nucleares/agonistas , beta-Lactamasas/química , Animales , Benzoatos/química , Benzoatos/farmacología , Bencilaminas/química , Bencilaminas/farmacología , Células CHO , Cricetinae , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.
Asunto(s)
Nanotecnología/métodos , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , beta-Lactamasas/metabolismo , Animales , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Estradiol/metabolismo , Estradiol/farmacología , Receptor beta de Estrógeno , Vectores Genéticos , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores de Estrógenos/genética , Factores de Transcripción/genética , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Endothelial lipase is a phospholipase with activity against high-density lipoprotein. Although a small number of mutations in LIPG have been described, the role of LIPG in protection against atherosclerosis is unclear. METHODS AND RESULTS: We identified 8 loss-of-function (LOF) mutations in LIPG in individuals with high-density lipoprotein cholesterol. Functional analysis confirmed that most rare mutations abolish lipase activity in vitro, indicating complete LOF, whereas 2 more common mutations N396S and R476W reduce activity by ≈50%, indicating partial LOF and implying ≈50% and ≈75% remaining endothelial lipase function in heterozygous complete LOF and partial LOF mutation carriers, respectively. complete LOF mutation carriers had significantly higher plasma high-density lipoprotein cholesterol levels compared with partial LOF mutation carriers. Apolipoprotein B-depleted serum from complete LOF carriers showed significantly enhanced cholesterol efflux acceptor capacity, whereas only trends were observed in partial LOF carriers. Carriers of LIPG mutations exhibited trends toward reduced coronary artery disease in 4 independent cohorts (meta-analysis odds ratio, 0.7; P=0.04). CONCLUSIONS: Our data suggest that the impact of LIPG mutations is directly related to their effect on endothelial lipase function and support that antagonism of endothelial lipase function improves cardioprotection.
Asunto(s)
HDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/enzimología , Enfermedad de la Arteria Coronaria/genética , Lipasa/genética , Mutación Missense , Estudios de Cohortes , Enfermedad de la Arteria Coronaria/metabolismo , Heterocigoto , Humanos , Lipasa/metabolismoRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.
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Anticuerpos Monoclonales/uso terapéutico , Anticolesterolemiantes/uso terapéutico , LDL-Colesterol/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Inmunización Pasiva , Síndrome Metabólico/terapia , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/inmunología , Serina Endopeptidasas/inmunología , Simvastatina/uso terapéutico , Proteínas de Unión a los Elementos Reguladores de Esteroles/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Anticolesterolemiantes/administración & dosificación , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/genética , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Perfilación de la Expresión Génica , Células Hep G2/efectos de los fármacos , Células Hep G2/metabolismo , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Macaca mulatta , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/genética , Ratones , Ratones Transgénicos , Proproteína Convertasa 9 , Proproteína Convertasas/biosíntesis , Proproteína Convertasas/genética , ARN Mensajero/metabolismo , Receptores de LDL/biosíntesis , Receptores de LDL/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Simvastatina/administración & dosificaciónRESUMEN
The association between hypercholesterolemia and elevated serum apolipoprotein B (APOB) has generated interest in APOB as a therapeutic target for patients at risk of developing cardiovascular disease. In the clinic, mipomersen, an antisense oligonucleotide (ASO) APOB inhibitor, was associated with a trend toward increased hepatic triglycerides, and liver steatosis remains a concern. We found that siRNA-mediated knockdown of ApoB led to elevated hepatic triglycerides and liver steatosis in mice engineered to exhibit a human-like lipid profile. Many genes required for fatty acid synthesis were reduced, suggesting that the observed elevation in hepatic triglycerides is maintained by the cell through fatty acid uptake as opposed to fatty acid synthesis. Fatty acid transport protein 5 (Fatp5/Slc27a5) is required for long chain fatty acid (LCFA) uptake and bile acid reconjugation by the liver. Fatp5 knockout mice exhibited lower levels of hepatic triglycerides due to decreased fatty acid uptake, and shRNA-mediated knockdown of Fatp5 protected mice from diet-induced liver steatosis. Here, we evaluated if siRNA-mediated knockdown of Fatp5 was sufficient to alleviate ApoB knockdown-induced steatosis. We determined that, although Fatp5 siRNA treatment was sufficient to increase the proportion of unconjugated bile acids 100-fold, consistent with FATP5's role in bile acid reconjugation, Fatp5 knockdown failed to influence the degree, zonal distribution, or composition of the hepatic triglycerides that accumulated following ApoB siRNA treatment.
Asunto(s)
Apolipoproteínas B/genética , Proteínas de Transporte de Ácidos Grasos/genética , Hígado Graso/genética , Animales , Apolipoproteínas B/sangre , Ácidos y Sales Biliares/metabolismo , Colesterol/sangre , HDL-Colesterol/sangre , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño , Triglicéridos/sangreRESUMEN
A high content molecular fragmentation for the analysis of phosphatidylcholines (PC) was achieved utilizing a two-stage [trap (first generation fragmentation) and transfer (second generation fragmentation)] collision-induced dissociation (CID) in combination with travelling-wave ion mobility spectrometry (TWIMS). The novel aspects of this work reside in the fact that a TWIMS arrangement was used to obtain a high level structural information including location of fatty acyl substituents and double bonds for PCs in plasma, and the presence of alkali metal adduct ions such as [M + Li](+) was not required to obtain double bond positions. Elemental compositions for fragment ions were confirmed by accurate mass measurements. A very specific first generation fragment ion m/z 577 (M-phosphoryl choline) from the PC [16:0/18:1 (9Z)] was produced, which by further CID generated acylium ions containing either the fatty acyl 16:0 (C(15)H(31)CO(+), m/z 239) or 18:1 (9Z) (C(17)H(33)CO(+), m/z 265) substituent. Subsequent water loss from these acylium ions was key in producing hydrocarbon fragment ions mainly from the α-proximal position of the carbonyl group such as the hydrocarbon ion m/z 67 (+H(2)C-HC = CH-CH = CH(2)). Formation of these ions was of important significance for determining double bonds in the fatty acyl chains. In addition to this, and with the aid of (13)C labeled lyso-phosphatidylcholine (LPC) 18:1 (9Z) in the ω-position (methyl) TAP fragmentation produced the ion at m/z 57. And was proven to be derived from the α-proximal (carboxylate) or distant ω-position (methyl) in the LPC.
Asunto(s)
Fosfatidilcolinas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Ceramidas/sangre , Ceramidas/química , Iones/química , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Peso Molecular , Fosfatidilcolinas/sangre , Fosfolípidos/sangre , Fosfolípidos/química , Esfingomielinas/sangre , Esfingomielinas/química , Agua/químicaRESUMEN
Insulin resistance is one of the dominant symptoms of type 2 diabetes (T2D). Although the molecular mechanisms leading to this resistance are largely unknown, experimental data support that the insulin signaling pathway is impaired in patients who are insulin resistant. To identify novel components/modulators of the insulin signaling pathway, we designed siRNAs targeting over 300 genes and tested the effects of knocking down these genes in an insulin-dependent, anti-lipolysis assay in 3T3-L1 adipocytes. For 126 genes, significant changes in free fatty acid release were observed. However, due to off-target effects (in addition to other limitations), high-throughput RNAi-based screens in cell-based systems generate significant amounts of noise. Therefore, to obtain a more reliable set of genes from the siRNA hits in our screen, we developed and applied a novel network-based approach that elucidates the mechanisms of action for the true positive siRNA hits. Our analysis results in the identification of a core network underlying the insulin signaling pathway that is more significantly enriched for genes previously associated with insulin resistance than the set of genes annotated in the KEGG database as belonging to the insulin signaling pathway. We experimentally validated one of the predictions, S1pr2, as a novel candidate gene for T2D.