RESUMEN
With the advent of high-throughput sequencing of immunoglobulin genes (Ig-Seq), the understanding of antibody repertoires and their dynamics among individuals and populations has become an exciting area of research. There is an increasing number of computational tools that aid in every step of the immune repertoire characterization. However, since not all tools function identically, every pipeline has its unique rationale and capabilities, creating a rich blend of useful features that may appear intimidating for newcomer laboratories with the desire to plunge into immune repertoire analysis to expand and improve their research; hence, all pipeline strengths and differences may not seem evident. In this review we provide a practical and organized list of the current set of computational tools, focusing on their most attractive features and differences in order to carry out the characterization of antibody repertoires so that the reader better decides a strategic approach for the experimental design, and computational pathways for the analyses of immune repertoires.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Programas Informáticos , Recombinación V(D)J , Animales , Humanos , InmunoglobulinasRESUMEN
Using C9 cells stably expressing LPA1 receptors fused to the enhanced green fluorescent protein, it was observed that activation of protein kinase C induced a rapid and strong increase in the phosphorylation state of these receptors. Overnight incubation with phorbol esters markedly decreased the amount of conventional (α, ßI, ßII and γ) and novel (δ) but not atypical (ζ) immunodetected PKC isoforms, this treatment blocks the action of protein kinase on receptor function and phosphorylation. Bis-indolylmaleimide I a general, non-subtype selective protein kinase C inhibitor, and Gö 6976, selective for the isoforms α and ß, were also able to block LPA1 receptor desensitization and phosphorylation; hispidin, isoform ß-selective blocker partially avoided receptor desensitization. Expression of dominant-negative protein kinase C α or ß II mutants and knocking down the expression of these kinase isozymes markedly decreased phorbol ester-induced LPA1 receptor phosphorylation without avoiding receptor desensitization. This effect was blocked by bis-indolyl-maleimide and Gö 6976, suggesting that these genetic interventions were not completely effective. It was also observed that protein kinase C α and ß II isozymes co-immunoprecipitate with LPA1 receptors and that such an association was further increased by cell treatments with phorbol esters or lysophosphatidic acid. Our data suggest that conventional protein kinase C α and ß isozymes modulate LPA1 receptor phosphorylation state. Receptor desensitization appears to be a more complex process that might involve additional elements.
Asunto(s)
Proteína Quinasa C/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Animales , Línea Celular , Isoenzimas/metabolismo , Lisofosfolípidos/farmacología , Fosforilación/efectos de los fármacos , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The effect of insulin-like growth factor-I (IGF-I) on human alpha(1B)-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human alpha(1B)-adrenoceptor. Receptors were identified by radioligand binding and photoaffinity labeling, and were immunoprecipitated with an antiserum generated against the enhanced green fluorescent protein. The receptor was functional, as evidenced by noradrenaline action on intracellular calcium and inositol phosphate production. IGF-I had no significant effect by itself on these parameters but markedly reduced the effects of noradrenaline. IGF-I induced alpha(1B)-adrenoceptor phosphorylation, which was markedly reduced by the following agents: pertussis toxin, a metalloproteinase inhibitor, diphtheria toxin mutant CRM 197, an epidermal growth factor (EGF) receptor intrinsic kinase activity inhibitor, and by phosphoinositide 3-kinase and protein kinase C inhibitors. IGF-I action appears to involve activation of a pertussis toxin-sensitive G protein, shedding of heparin-binding EGF and autocrine activation of EGF receptors. G protein subunits and phosphotyrosine residues stimulate phosphoinositide 3-kinase activity leading to activation of protein kinase C, which in turn phosphorylates alpha(1B)-adrenoceptors. Confocal fluorescent microscopy showed that alpha(1B)-adrenoceptors fussed to the green fluorescent protein were located in plasma membrane and intracellular vesicles in the basal state. IGF-I induced receptor redistribution favoring the intracellular location; this effect was blocked by hypertonic sucrose and concanavalin A. Our data show that IGF-I induces alpha(1B)-adrenoceptor desensitization associated to receptor phosphorylation and internalization.