RESUMEN
Circulating proteins are vital in human health and disease and are frequently used as biomarkers for clinical decision-making or as targets for pharmacological intervention. Here, we map and replicate protein quantitative trait loci (pQTL) for 90 cardiovascular proteins in over 30,000 individuals, resulting in 451 pQTLs for 85 proteins. For each protein, we further perform pathway mapping to obtain trans-pQTL gene and regulatory designations. We substantiate these regulatory findings with orthogonal evidence for trans-pQTLs using mouse knockdown experiments (ABCA1 and TRIB1) and clinical trial results (chemokine receptors CCR2 and CCR5), with consistent regulation. Finally, we evaluate known drug targets, and suggest new target candidates or repositioning opportunities using Mendelian randomization. This identifies 11 proteins with causal evidence of involvement in human disease that have not previously been targeted, including EGF, IL-16, PAPPA, SPON1, F3, ADM, CASP-8, CHI3L1, CXCL16, GDF15 and MMP-12. Taken together, these findings demonstrate the utility of large-scale mapping of the genetics of the proteome and provide a resource for future precision studies of circulating proteins in human health.
Asunto(s)
Sistema Cardiovascular/metabolismo , Mapeo Cromosómico , Sistemas de Liberación de Medicamentos , Genómica , Transportador 1 de Casete de Unión a ATP/genética , Asma/genética , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Humanos , Enfermedades Inflamatorias del Intestino/genética , Proteína 1 Similar al Receptor de Interleucina-1/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Desequilibrio de Ligamiento , Análisis de la Aleatorización Mendeliana , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteoma , Sitios de Carácter Cuantitativo , Receptores CCR2/genética , Receptores CCR5/genéticaRESUMEN
PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1ß production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Animales , Artritis Experimental/inmunología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Interleucina-10/inmunología , Interleucina-1beta/inmunología , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 3/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirroles/farmacocinética , Pirroles/farmacología , Ratas , Células TH1/citología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th17/citología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
PURPOSE: Growth and differentiation factor 8 (GDF-8) is a negative regulator of skeletal muscle mass and targeted by inhibitors to treat diseases associated with muscle loss. In order to enable clinical and translational investigations of GDF-8 inhibitors, specific and sensitive measurements of GDF-8 are necessary. EXPERIMENTAL DESIGN: An immunoaffinity LC-MS/MS assay for quantification of GDF-8 in serum was developed, qualified and implemented. The workflow includes offline enrichment of GDF-8 using an anti-GDF-8 antibody, followed by isolation using magnetic beads, trypsin digestion, and quantification using 2D nanoflow LC-MS/MS. RESULTS: This assay was qualified in human serum with a lower LOQ of 1.0 ng/mL based on the intact protein. GDF-8 was quantified in serum from juvenile and adult humans as well as mouse, rat, and cynomolgus monkey. Additionally, the assay was utilized to demonstrate an increase of total GDF-8 in serum following administration of an anti-GDF-8 monoclonal antibody therapeutic in cynomolgus monkeys. CONCLUSIONS AND CLINICAL RELEVANCE: A specific and sensitive method was developed for the measurement of GDF-8 in juvenile and adult serum samples as well as preclinical species. The confident quantification of GDF-8 now enables a greater understanding of any association between changes GDF-8 levels and muscle mass.
Asunto(s)
Anticuerpos Monoclonales/química , Bioensayo/normas , Miostatina/sangre , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Expresión Génica , Humanos , Separación Inmunomagnética/métodos , Macaca fascicularis , Ratones , Miostatina/genética , Péptidos/síntesis química , Ratas , Espectrometría de Masas en Tándem/métodosRESUMEN
The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34-38 kDa. Its polypeptide monomer contains one canonical N-glycosylation site at Asn68, and human recombinant IL-17A was partly N-glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N-glycopeptide 63-69 indicated that the N-glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N-glycosylated polypeptides. These were caused by unpredicted partial O-glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O-glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N-glycosylation on recombinant IL-17A but additionally created an O-glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N-glycosylation on the product, but this work exemplifies how a host may also unpredictably O-glycosylate a protein that is probably not normally O-glycosylated.
Asunto(s)
Interleucina-17/biosíntesis , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilación , Células HEK293 , Humanos , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/química , Interleucina-17/química , Meliteno/biosíntesis , Meliteno/química , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Señales de Clasificación de Proteína , Proteínas Recombinantes de Fusión/química , Espectrometría de Masas en TándemRESUMEN
The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.
Asunto(s)
Asma/enzimología , Quitinasas/antagonistas & inhibidores , Hipersensibilidad/enzimología , Alérgenos/inmunología , Animales , Asma/genética , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Quitinasas/deficiencia , Quitinasas/genética , Quitinasas/inmunología , Femenino , Humanos , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Inflamación/enzimología , Inflamación/genética , Inflamación/inmunología , Interleucina-13/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Neutrófilos/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K(m) of 28 microM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization.
Asunto(s)
Amidohidrolasas/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Inhibidores Enzimáticos/química , Espectrometría de Masas/métodos , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/química , Proteínas Ligadas a GPI , Ensayos Analíticos de Alto Rendimiento , Humanos , Riñón/enzimología , Ratones , Datos de Secuencia Molecular , Ácido Pantoténico/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría UltravioletaRESUMEN
IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica/inmunología , Interleucina-17/inmunología , Activación de Linfocitos/inmunología , Secuencias de Aminoácidos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células CHO , Cricetinae , Cricetulus , Cisteína/genética , Cisteína/inmunología , Dimerización , Expresión Génica , Regulación de la Expresión Génica/genética , Humanos , Inmunidad Celular , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucina-17/farmacología , Activación de Linfocitos/efectos de los fármacosRESUMEN
A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.