RESUMO
Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a, also known as Stem Cell Ag-1 and T cell-activating protein, is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1-expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S, which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.
Assuntos
Baço , Viroses , Animais , Antígenos Ly/genética , Humanos , Inflamação/genética , Linfócitos , Proteínas de Membrana/genética , Camundongos , Família Multigênica , Viroses/genéticaRESUMO
Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. IMPORTANCE Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV's genome encodes 10 major proteins, 9 of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here, we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein, a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.
Assuntos
Nucleoproteínas , Vírus de RNA , Tilápia , Animais , Doenças dos Peixes/virologia , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Infecções por Vírus de RNA/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/patogenicidade , RNA Viral/genética , Tilápia/genéticaRESUMO
Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Imunotoxinas/imunologia , Mucina-1/química , Mucina-1/imunologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Domínios Proteicos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Membrana Celular/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Proteína Morfogenética Óssea 2/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Glicosilação/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Dobramento de Proteína/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program.The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE-green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citoplasma/metabolismo , Proteínas Repressoras/metabolismo , Cromatina/metabolismo , Cromatografia em Gel , Imunoprecipitação , Microscopia Confocal , Plantas Geneticamente Modificadas , Complexo Repressor Polycomb 2RESUMO
Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described.
Assuntos
Núcleo Celular/metabolismo , Mucina-1/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Códon , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Mucina-1/metabolismo , Neoplasias Pancreáticas/metabolismoRESUMO
Immunotherapy using mucin 1 (MUC1) linked to oxidised mannan (MFP) was investigated in an aggressive MUC1+ metastatic tumour, DA3-MUC1 because, unlike many MUC1+ tumour models, DA3-MUC1 is not spontaneously rejected in mice making it an alternative model for immunotherapy studies. Further, DA3-MUC1 cells are resistant to lysis by anti-MUC1 cytotoxic T cells (CTLs). The inability of DA3-MUC1 tumours to be rejected in naïve mice as well as vaccination to MUC1 was attributed to a deficiency of expression of MHC class I molecules on the tumour cell surface. In vitro and in vivo analysis of subcutaneous tumours and lung metastases demonstrated that DA3-MUC1 tumour cells have a low expression (< 6%) of MHC class I which can be upregulated (> 90%) following culturing with IFN-γ. Results from flow cytometry analysis and immunoperoxidase staining indicated that the in vitro up-regulation of MHC class I could be maintained for up to seven days in vivo, without affecting the expression levels of MUC1 antigen. Interestingly, MUC1-specific CTL that lyse DA3-MUC1 targets in vitro were induced in MFP immunised mice but failed to protect mice from a DA3-MUC1 tumour challenge. These results highlight the importance of MHC class I molecules in the induction of anti-tumour immunity and the MFP immune response.
Assuntos
Adenocarcinoma/etiologia , Adenocarcinoma/secundário , Neoplasias da Mama/patologia , Imunoterapia , Interferon gama/fisiologia , Mucina-1/imunologia , Animais , Neoplasias da Mama/etiologia , Feminino , Antígenos de Histocompatibilidade Classe I , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Neurogenesis and neuronal recruitment occur in adult brains of many vertebrates, and the hypothesis is that these phenomena contribute to the brain plasticity that enables organisms to adjust to environmental changes. In mammals, vasoactive intestinal polypeptide (VIP) is known to have many neuroprotective properties, but in the avian brain, although widely distributed, its role in neuronal recruitment is not yet understood. In the present study we actively immunized adult zebra finches against VIP conjugated to KLH and compared neuronal recruitment in their brains, with brains of control birds, which were immunized against KLH. We looked at two forebrain regions: the nidopallium caudale (NC), which plays a role in vocal communication, and the hippocampus (HC), which is involved in the processing of spatial information. Our data demonstrate that active immunization against VIP reduces neuronal recruitment, inhibits reproduction, and induces molting, with no change in plasma prolactin levels. Thus, our observations suggest that VIP has a direct positive role in neuronal recruitment and reproduction in birds. J. Comp. Neurol. 524:2516-2528, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Neurogênese/fisiologia , Neurônios/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Vacinação/métodos , Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Sequência de Aminoácidos/fisiologia , Animais , Galinhas , Feminino , Tentilhões , Neurogênese/efeitos dos fármacos , Neurônios/fisiologia , Reprodução/fisiologia , Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
The levels and intracellular localization of wild-type transforming growth factor ß superfamily (TGFß-SF) receptors are tightly regulated by endocytic trafficking, shedding and degradation. In contrast, a main regulatory mechanism of mutation-bearing receptors involves their intracellular retention. Anti-Müllerian hormone receptor II (AMHRII, also known as AMHR2) is the type-II receptor for anti-Müllerian hormone (AMH), a TGFß-SF ligand that mediates Müllerian duct regression in males. Here, we studied AMHRII processing and identified novel mechanisms of its constitutive negative regulation. Immunoblot analysis revealed that a significant portion of AMHRII was missing most of its extracellular domain (ECD) and, although glycosylated, was unfolded and retained in the endoplasmic reticulum. Exogenous expression of AMHRII, but not of type-II TGF-ß receptor (TßRII, also known as TGFR2), resulted in its disulfide-bond-mediated homo-oligomerization and intracellular retention, and in a decrease in its AMH-binding capacity. At the plasma membrane, AMHRII differed from TßRII, forming high levels of non-covalent homomeric complexes, which exhibited a clustered distribution and restricted lateral mobility. This study identifies novel mechanisms of negative regulation of a type-II TGFß-SF receptor through cleavage, intracellular retention and/or promiscuous disulfide-bond mediated homo-oligomerization.
Assuntos
Processamento de Proteína Pós-Traducional , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Hormônio Antimülleriano/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Ligação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transforming growth factor-ß (TGF-ß) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context-dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-ß signaling. However, the molecular mechanism(s) involved in the regulation of TGF-ß signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-ß receptor (TßRI) with TGF-ß phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TßRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA-mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-ß-induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-ß in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TßRI in the membrane, balancing TGF-ß signaling via the Smad and JNK pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colesterol/metabolismo , Endocitose/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Clatrina , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genéticaRESUMO
The MUC1 tumor associated antigen is highly expressed on a range of tumors. Its broad distribution on primary tumors and metastases renders it an attractive target for immunotherapy. After synthesis MUC1 is cleaved, yielding a large soluble extracellular alpha subunit containing the tandem repeats array (TRA) domain specifically bound, via non-covalent interaction, to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Thus far, inconclusive efficacy has been reported for anti-MUC1 antibodies directed against the soluble alpha subunit. Targeting the cell bound beta subunit, may bypass limitations posed by circulating TRA domains. MUC1's signal peptide (SP) domain promiscuously binds multiple MHC class II and Class I alleles, which upon vaccination, generated robust T-cell immunity against MUC1-positive tumors. This is a first demonstration of non-MHC associated, MUC1 specific, cell surfaces presence for MUC1 SP domain. Polyclonal and monoclonal antibodies generated against MUC1 SP domain specifically bind a large variety of MUC1-positive human solid and haematological tumor cell lines; MUC1-positive bone marrow derived plasma cells obtained from multiple myeloma (MM)-patients, but not MUC1 negative tumors cells, and normal naive primary blood and epithelial cells. Membranal MUC1 SP appears mainly as an independent entity but also co-localized with the full MUC1 molecule. MUC1-SP specific binding in BM-derived plasma cells can assist in selecting patients to be treated with anti-MUC1 SP therapeutic vaccine, ImMucin. A therapeutic potential of the anti-MUC1 SP antibodies was suggested by their ability to support of complement-mediated lysis of MUC1-positive tumor cells but not MUC1 negative tumor cells and normal naive primary epithelial cells. These findings suggest a novel cell surface presence of MUC1 SP domain, a potential therapeutic benefit for anti-MUC1 SP antibodies in MUC1-positive tumors and a selection tool for MM patients to be treated with the anti-MUC1 SP vaccine, ImMucin.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Vacinas Anticâncer/administração & dosagem , Mucina-1/imunologia , Mieloma Múltiplo/tratamento farmacológico , Peptídeos/imunologia , Subunidades Proteicas/imunologia , Idoso , Animais , Anticorpos Monoclonais/biossíntese , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Imunidade Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mucina-1/química , Mucina-1/genética , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Peptídeos/administração & dosagem , Peptídeos/síntese química , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/patologia , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Coelhos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.
Assuntos
Apoptose , Autofagia , Doenças dos Bovinos/fisiopatologia , Vírus da Doença Hemorrágica Epizoótica/fisiologia , Infecções por Reoviridae/veterinária , Doenças dos Ovinos/fisiopatologia , Animais , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/virologia , Linhagem Celular , Vírus da Doença Hemorrágica Epizoótica/genética , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/fisiopatologia , Infecções por Reoviridae/virologia , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/metabolismo , Doenças dos Ovinos/virologia , Transdução de SinaisRESUMO
The low protection by the bacillus Calmette-Guérin (BCG) vaccine and existence of drug-resistant strains require better anti-Mycobacterium tuberculosis vaccines with a broad, long-lasting, antigen-specific response. Using bioinformatics tools, we identified five 19- to 40-mer signal peptide (SP) domain vaccine candidates (VCs) derived from M. tuberculosis antigens. All VCs were predicted to have promiscuous binding to major histocompatibility complex (MHC) class I and II alleles in large geographic territories worldwide. Peripheral mononuclear cells (PBMC) from healthy naïve donors and tuberculosis patients exhibited strong proliferation that correlated positively with Th1 cytokine secretion only in healthy naïve donors. Proliferation to SP VCs was superior to that to antigen-matched control peptides with similar length and various MHC class I and II binding properties. T-cell lines induced to SP VCs from healthy naïve donors had increased CD44(high)/CD62L(+) activation/effector memory markers and gamma interferon (IFN-γ), but not interleukin-4 (IL-4), production in both CD4(+) and CD8(+) T-cell subpopulations. T-cell lines from healthy naïve donors and tuberculosis patients also manifested strong, dose-dependent, antigen-specific cytotoxicity against autologous VC-loaded or M. tuberculosis-infected macrophages. Lysis of M. tuberculosis-infected targets was accompanied by high IFN-γ secretion. Various combinations of these five VCs manifested synergic proliferation of PBMC from selected healthy naïve donors. Immunogenicity of the best three combinations, termed Mix1, Mix2, and Mix3 and consisting of 2 to 5 of the VCs, was then evaluated in mice. Each mixture manifested strong cytotoxicity against M. tuberculosis-infected macrophages, while Mix3 also manifested a VC-specific humoral immune response. Based on these results, we plan to evaluate the protection properties of these combinations as an improved tuberculosis subunit vaccine.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Animais , Proliferação de Células , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Subpopulações de Linfócitos T/imunologia , Vacinas contra a Tuberculose/administração & dosagemRESUMO
Naturally generated autoantibodies to tumor-associated antigens such as MUC1 can assist in cancer diagnosis and prognosis. While previous studies have concentrated on the tandem repeat array domain of MUC1, here we focused on MUC1's signal peptide domain. We used ELISA assays with MUC1-specific epitopes and antibodies to quantify soluble MUC1 antigen and anti-MUC1 autoantibodies against the tandem repeat array and signal peptide domains in 15 naïve donors and 27 multiple myeloma cancer patients. We showed a significant increase in up to 24-fold (P<0.004) only in the levels of anti-MUC1 signal peptide autoantibodies in the sera of multiple myeloma patients vs. naïve donors. This increase stemmed chiefly from the preferred immunogenicity of the signal peptide. Moreover, a significant positive correlation (R(2)=0.5361, P<0.048, Pearson correlation) was shown between the levels of soluble MUC1 and anti-MUC1 signal peptide autoantibodies in multiple myeloma patients with progressive disease while under therapy. This is an initial report on the existence of autoantibodies to a signal peptide domain in general and to the MUC1 signal peptide domain in particular in cancer patients. The autoantibodies had MUC1 rather than signal peptide specificity. The specific nature of the antigen leading to generation of these autoantibodies is still unclear because it is unlikely that the target antigen is a major histo-compatibility complex-peptide complex and we could not trace soluble MUC1 signal peptide fragments in naïve donors and multiple myeloma patients. Further validation of these findings may improve diagnostic and prognostic capabilities for MUC1-positive multiple myeloma patients and potentially, patients with other MUC1-positive cancers, as well.
RESUMO
The response to transforming growth factor-ß (TGF-ß) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-ß receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-ß stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-ß receptor (TßRII). Moreover, following the stimulation of mitotic cells with TGF-ß, the proteasome-mediated attenuation of TGF-ß receptor activity, the degradation and clearance of TßRII from the plasma membrane, and the clearance of the TGF-ß ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-ß receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-ß stimuli in mitosis.
Assuntos
Mitose , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Feminino , Humanos , Ligantes , Mesoderma/patologia , Mitose/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The cell-surface glycoprotein MUC1 is a particularly appealing target for antibody targeting, being selectively overexpressed in many types of cancers and a high proportion of cancer stem-like cells. However the occurrence of MUC1 cleavage, which leads to the release of the extracellular α subunit into the circulation where it can sequester many anti-MUC1 antibodies, renders the target problematic to some degree. To address this issue, we generated a set of unique MUC1 monoclonal antibodies that target a region termed the SEA domain that remains tethered to the cell surface after MUC1 cleavage. In breast cancer cell populations, these antibodies bound the cancer cells with high picomolar affinity. Starting with a partially humanized antibody, DMB5F3, we created a recombinant chimeric antibody that bound a panel of MUC1+ cancer cells with higher affinities relative to cetuximab (anti-EGFR1) or tratuzumab (anti-erbB2) control antibodies. DMB5F3 internalization from the cell surface occurred in an efficient temperature-dependent manner. Linkage to toxin rendered these DMB5F3 antibodies to be cytotoxic against MUC1+ cancer cells at low picomolar concentrations. Our findings show that high-affinity antibodies to cell-bound MUC1 SEA domain exert specific cytotoxicity against cancer cells, and they point to the SEA domain as a potential immunogen to generate MUC1 vaccines.
Assuntos
Anticorpos Monoclonais/imunologia , Mucina-1/imunologia , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , HumanosRESUMO
We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.
Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência , Biossíntese de Proteínas , RNA de Transferência , Análise de Célula Única , Animais , Astrócitos/metabolismo , Células CHO , Cricetinae , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/análise , Camundongos , Camundongos Endogâmicos C57BL , RNA de Transferência/análise , Proteínas Virais/biossínteseRESUMO
An optimal cancer vaccine should be able to induce highly potent, long-lasting, tumor-specific responses in the majority of the cancer patient population. One approach for achieving this is to use synthetic peptide vaccines derived from widely expressed tumor-associated antigens, that promiscuously bind multiple MHC class I and class II alleles. MUC1-SP-L (ImMucin, VXL100) is a 21mer peptide encoding the complete signal peptide domain of MUC1, a tumor-associated antigen expressed by over 90% of solid and non-solid tumors. MUC1-SP-L was predicted in silico to bind various MHC class I and MHC class II alleles, covering the majority of the Caucasian population. PBLs obtained from 13 naïve donors all proliferated, with a Stimulation Index (SI≥2), to the MUC1-SP-L peptide, producing mixed CD4+ and CD8+ responses. Similar results were manifested by MUC1-SP-L in PBLs derived from 9 of 10 cancer patients with MUC1 positive tumors. CD4+ and CD8+ T cell populations exhibited CD45RO memory markers and secreted IFN-gamma and IL-2 following stimulation with MUC1-SP-L. These T cells also exhibited proliferation to the MUC1-SP-L inner 9mer epitopes and cytotoxicity against tumor cell lines expressing MUC1 and a concordant MHC class I allele. Cytotoxicity to MUC1-expressing human and murine tumors was shown also in T cells obtained from HLA-A2 transgenic mice and BALB/c syngeneic mice immunized with the MUC1-SP-L and GM-CSF. In an immunotherapy model, BALB/c mice inoculated with metastatic MUC1 transfected murine DA3 mammary tumor cells, exhibited significantly prolonged survival following vaccination with MUC1-SP-L. Our results indicate superior immunological and anti-tumor properties of MUC1-SP-L compared to previously published MUC1-derived epitopes.
Assuntos
Vacinas Anticâncer/administração & dosagem , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Imunoterapia/métodos , Mucina-1/biossíntese , Mucina-1/imunologia , Neoplasias/terapia , Idoso , Animais , Vacinas Anticâncer/imunologia , Proliferação de Células , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pessoa de Meia-Idade , Ligação Proteica , Doenças dos Roedores/terapia , Análise de SobrevidaRESUMO
The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/química , Proteínas Mutadas de Ataxia Telangiectasia , Cromatina/química , Cromatina/metabolismo , Ensaio Cometa/métodos , Células HeLa , Histonas/química , Humanos , Cinética , Fosforilação , Processamento de Proteína Pós-Traducional , Interferência de RNA , Recombinação Genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle.