RESUMEN
Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple beta2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial beta2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. beta2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling beta2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.
Asunto(s)
Antígenos CD18/fisiología , Proteínas de Ciclo Celular , Factores de Intercambio de Guanina Nucleótido/fisiología , Neutrófilos/fisiología , Animales , Adhesión Celular , Quimiotaxis de Leucocito , Endotelio Vascular/citología , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Ratones Noqueados , Neutrófilos/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/fisiología , Fagocitosis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-vav , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Immune responses targeting self-proteins (autoantigens) can lead to a variety of autoimmune diseases. Identification of these antigens is important for both diagnostic and therapeutic reasons. However, current approaches to characterize autoantigens have, in most cases, met only with limited success. Here we present a synthetic representation of the complete human proteome, the T7 peptidome phage display library (T7-Pep), and demonstrate its application to autoantigen discovery. T7-Pep is composed of >413,000 36-residue, overlapping peptides that cover all open reading frames in the human genome, and can be analyzed using high-throughput DNA sequencing. We developed a phage immunoprecipitation sequencing (PhIP-Seq) methodology to identify known and previously unreported autoantibodies contained in the spinal fluid of three individuals with paraneoplastic neurological syndromes. We also show how T7-Pep can be used more generally to identify peptide-protein interactions, suggesting the broader utility of our approach for proteomic research.
Asunto(s)
Autoantígenos/inmunología , Autoantígenos/aislamiento & purificación , Biblioteca de Péptidos , Proteoma/genética , Proteómica/métodos , Secuencia de Aminoácidos , Antígenos de Neoplasias/inmunología , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Autoantígenos/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Bacteriófago T7/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Clonación Molecular , Femenino , Biblioteca de Genes , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoprecipitación , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/inmunología , Antígeno Ventral Neuro-Oncológico , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta/inmunología , Síndromes Paraneoplásicos del Sistema Nervioso/inmunología , Proteínas de Unión al ARN/inmunología , Análisis de Secuencia de ARNRESUMEN
Vav guanine nucleotide exchange factors (GEFs) have been implicated in cell adhesion by integrin and immune response receptors through the regulation of Rho GTPases. Here, we examine the role of Vav and Rho GTPases in phagocytosis by using primary murine macrophages. The genetic deletion of Rac1 and Rac2 prevents phagocytosis mediated by integrin and Fcgamma receptors (FcgammaR), whereas the genetic deletion of Vav1 and Vav3 only prevents integrin-mediated phagocytosis through the complement receptor alpha(M)beta(2). In addition, a Rac1/2 or Vav1/3 deficiency blocks Arp2/3 recruitment and actin polymerization at the complement-induced phagosome, indicating that these proteins regulate early steps in phagocytosis. Moreover, constitutively active Rac is able to rescue actin polymerization and complement-mediated phagocytosis in Vav-deficient macrophages. These studies indicate that Rac is critical for complement- and FcgammaR-mediated phagocytosis. In contrast, Vav is specifically required for complement-mediated phagocytosis, suggesting that Rac is regulated by GEFs other than Vav downstream of the FcgammaR.