RESUMEN
The human inhibitor of Bruton's tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3(IBTK) complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3(IBTK) for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3(IBTK) regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5'-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3(IBTK) as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Glutatión/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lentivirus/metabolismo , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Homología de Secuencia de Ácido Nucleico , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection. Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex. Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity. The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity. Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2. Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.
Asunto(s)
VIH-1/fisiología , Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Unión Competitiva , Línea Celular , Células Cultivadas , Quimiocina CCL3/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Células HeLa , Humanos , Ratones , Monocitos/metabolismo , Monocitos/virología , Inhibidor NF-kappaB alfa , Activación TranscripcionalRESUMEN
The inhibitor of Bruton tyrosine kinase γ (IBtkγ) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtkγ-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtkγ at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtkγ-S87 and -S90 as the critical amino acid residues that regulate the IBtkγ binding affinity to Btk. Consistently, the mutants IBtkγ carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca(2+) fluxes and NF-κB activation on BCR triggering. Accordingly, spleen B cells from Ibtkγ(-/-) mice showed an increased activation of Btk, as evaluated by Y551-phosphorylation and sustained Ca(2+) mobilization on BCR engagement. These findings identify a novel pathway of Btk regulation via protein kinase C phosphorylation of IBtkγ.
Asunto(s)
Proteínas Portadoras/metabolismo , Células/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Agammaglobulinemia Tirosina Quinasa , Alanina/genética , Sustitución de Aminoácidos/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Mutación Missense/fisiología , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Serina/genética , Transducción de Señal/fisiologíaRESUMEN
The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100-120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis.
Asunto(s)
Antígenos de Neoplasias/química , Leucosialina/química , Acetilgalactosamina/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Electroforesis en Gel Bidimensional , Epítopos , Femenino , Glicosilación , Humanos , Leucosialina/genética , Leucosialina/inmunología , Leucosialina/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Interferencia de ARN , Espectrometría de Masas en TándemRESUMEN
B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Inmunoterapia/métodos , Linfoma de Células B/inmunología , Péptidos/inmunología , Animales , Anticuerpos Antiidiotipos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Idiotipos de Inmunoglobulinas/inmunología , Linfoma de Células B/terapia , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Biblioteca de Péptidos , Péptidos/uso terapéutico , Tomografía de Emisión de Positrones , Receptores de Antígenos de Linfocitos B/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.
Asunto(s)
Vacunas contra el SIDA/química , Epítopos/química , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/virología , VIH-1/química , Péptidos/química , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Animales , Epítopos/administración & dosificación , Epítopos/inmunología , Femenino , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/administración & dosificación , Péptidos/inmunología , Estructura Terciaria de Proteína , Conejos , Alineación de SecuenciaRESUMEN
The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5 min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30 s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/inmunología , Inhibidor NF-kappaB alfa/inmunología , FN-kappa B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Fosforilación , Transducción de SeñalRESUMEN
Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4(+) and CD8(+) T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.
Asunto(s)
Diferenciación Celular , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Análisis por Conglomerados , Citocinas/sangre , Citocinas/genética , Citocinas/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Lipopolisacáridos/inmunología , Recuento de Linfocitos , Depleción Linfocítica , Linfopoyesis , Ratones , Ratones Transgénicos , MicroARNs/genética , FN-kappa B/metabolismo , Subgrupos de Linfocitos T/metabolismo , Timocitos/citología , Timocitos/inmunología , Timo/citología , Timo/inmunología , Timo/metabolismoRESUMEN
Glycosylation is a posttranslational modification of proteins playing a major role in cell signalling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure variability and binding specificity to lectin ligands. Aberrant expression of glycan structures as well as occurrence of truncated structures, precursors, or novel structures of glycan may affect ligand-receptor interactions and thus interfere with regulation of cell adhesion, migration, and proliferation. Indeed, aberrant glycosylation represents a hallmark of cancer, reflecting cancer-specific changes in glycan biosynthesis pathways such as the altered expression of glycosyltransferases and glycosidases. Most studies have been carried out to identify changes in serum glycan structures. In most cancers, fucosylation and sialylation are significantly modified. Thus, aberrations in glycan structures can be used as targets to improve existing serum cancer biomarkers. The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. In this review, we discuss the aberrant protein glycosylation associated with human cancer and the identification of protein glycoforms as cancer biomarkers. In particular, we will focus on the aberrant CD43 glycosylation as cancer biomarker and the potential to exploit the UN1 monoclonal antibody (UN1 mAb) to identify aberrant CD43 glycoforms.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Leucosialina/metabolismo , Neoplasias/metabolismo , Animales , Biomarcadores de Tumor/sangre , Glicosilación , Humanos , Mucinas/metabolismo , Neoplasias/sangre , Polisacáridos/metabolismoRESUMEN
CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Inmunoterapia , Leucosialina/inmunología , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Epítopos/inmunología , Humanos , Leucosialina/genética , Ratones , Neoplasias/terapia , Células Tumorales CultivadasRESUMEN
Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.