RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Inmunotoxinas/inmunología , Mucina-1/química , Mucina-1/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Dominios Proteicos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina/química , Proteínas de la Ataxia Telangiectasia Mutada , Cromatina/química , Cromatina/metabolismo , Ensayo Cometa/métodos , Células HeLa , Histonas/química , Humanos , Cinética , Fosforilación , Procesamiento Proteico-Postraduccional , Interferencia de ARN , Recombinación Genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Membrana Celular/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Proteína Morfogenética Ósea 2/farmacología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Glicosilación/efectos de los fármacos , Humanos , Invasividad Neoplásica , Pliegue de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Procesamiento Proteico-Postraduccional , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Hormona Antimülleriana/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Unión Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Péptidos/química , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citoplasma/metabolismo , Proteínas Represoras/metabolismo , Cromatina/metabolismo , Cromatografía en Gel , Inmunoprecipitación , Microscopía Confocal , Plantas Modificadas Genéticamente , Complejo Represivo Polycomb 2RESUMEN
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.
Asunto(s)
Apoptosis , Autofagia , Enfermedades de los Bovinos/fisiopatología , Virus de la Enfermedad Hemorrágica Epizoótica/fisiología , Infecciones por Reoviridae/veterinaria , Enfermedades de las Ovejas/fisiopatología , Animales , Bovinos , Enfermedades de los Bovinos/genética , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/virología , Línea Celular , Virus de la Enfermedad Hemorrágica Epizoótica/genética , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Infecciones por Reoviridae/metabolismo , Infecciones por Reoviridae/fisiopatología , Infecciones por Reoviridae/virología , Ovinos , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/metabolismo , Enfermedades de las Ovejas/virología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Colorantes Fluorescentes , Microscopía Fluorescente , Biosíntesis de Proteínas , ARN de Transferencia , Análisis de la Célula Individual , Animales , Astrocitos/metabolismo , Células CHO , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/análisis , Ratones , Ratones Endogámicos C57BL , ARN de Transferencia/análisis , Proteínas Virales/biosíntesisRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Clatrina/metabolismo , Mitosis/fisiología , 2-Metoxiestradiol , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Clatrina/genética , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Estradiol/análogos & derivados , Estradiol/farmacología , Humanos , Mitosis/efectos de los fármacos , Mutación Missense , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Moduladores de Tubulina/farmacología , Proteínas Supresoras de TumorRESUMEN
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.
Asunto(s)
Bazo , Virosis , Animales , Antígenos Ly/genética , Humanos , Inflamación/genética , Linfocitos , Proteínas de la Membrana/genética , Ratones , Familia de Multigenes , Virosis/genéticaRESUMEN
Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Células Epiteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Eliminación de Gen , Mesodermo/citología , Mucina-1/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Secuencia de Bases , Línea Celular , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Immunoblotting , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Datos de Secuencia Molecular , Mucina-1/genéticaRESUMEN
MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating alpha and beta subunits, which specifically bind in a noncovalent interaction. Although the beta chain remains on the cell surface, the alpha chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic alpha-chain tandem repeat. Because the alpha-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 alpha/beta junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 alpha/beta junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 alpha/beta junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumor-suppressive activity. The MUC1/X alpha/beta junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound alpha/beta junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti alpha/beta junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound alpha/beta junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 alpha/beta junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies.
Asunto(s)
Inmunoterapia/métodos , Inmunotoxinas/química , Mucina-1/fisiología , Animales , Anticuerpos Monoclonales/química , Epítopos/química , Femenino , Humanos , Hibridomas/metabolismo , Ratones , Ratones SCID , Mucina-1/metabolismo , Trasplante de Neoplasias , Conformación Proteica , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.
Asunto(s)
Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Hidroliasas/fisiología , Antígeno CA-19-9 , Adhesión Celular/fisiología , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , ADN Complementario/genética , Regulación hacia Abajo , Selectina E/metabolismo , Endotelio Vascular/citología , Fucosa/metabolismo , Gangliósidos/biosíntesis , Gangliósidos/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Metástasis de la Neoplasia , Polisacáridos/biosíntesis , Polisacáridos/metabolismo , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Mucina-1/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Codón , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Ratones , Mucina-1/metabolismo , Neoplasias Pancreáticas/metabolismoRESUMEN
Cellular responses to DNA damage are mediated by an extensive network of signaling pathways. The ATM protein kinase is a master regulator of the response to double-strand breaks (DSBs), the most cytotoxic DNA lesion caused by ionizing radiation. ATM is the protein missing or inactive in patients with the pleiotropic genetic disorder ataxia-telangiectasia (A-T). A major response to DNA damage is altered expression of numerous genes. While studying gene expression in control and A-T cells following treatment with the radiomimetic chemical neocarzinostatin (NCS), we identified an expressed sequence tag that represented a gene that was induced by DSBs in an ATM-dependent manner. The corresponding cDNA encoded a dual specificity phosphatase of the MAP kinase phosphatase family, MKP-5. MKP-5 dephosphorylates and inactivates the stress-activated MAP kinases JNK and p38. The phosphorylation-dephosphorylation cycle of JNK and p38 by NCS was attenuated in A-T cells. Thus, ATM modulates this cycle in response to DSBs. These results further highlight ATM as a link between the DNA damage response and major signaling pathways involved in proliferative and apoptotic processes.
Asunto(s)
Daño del ADN , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Fosfatasas/biosíntesis , Activación Transcripcional , Cinostatina/farmacología , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Línea Celular , Proteínas de Unión al ADN , Fosfatasas de Especificidad Dual , Activación Enzimática , Eliminación de Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/biosíntesis , Radiación Ionizante , Proteínas Supresoras de Tumor , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.
Asunto(s)
Carcinoma/química , Neoplasias Endometriales/química , Endometrio/química , Trompas Uterinas/química , Mucina-1/química , Carcinoma/patología , Adhesión Celular , Polaridad Celular , Células Cultivadas/química , ADN Complementario/genética , Neoplasias Endometriales/patología , Células Epiteliales/química , Femenino , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Mucina-1/fisiología , Proteínas de Neoplasias/química , Isoformas de Proteínas/química , Estructura Terciaria de Proteína , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Vacunas contra el Cáncer/administración & dosificación , Mucina-1/inmunología , Mieloma Múltiple/tratamiento farmacológico , Péptidos/inmunología , Subunidades de Proteína/inmunología , Anciano , Animales , Anticuerpos Monoclonales/biosíntesis , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Línea Celular Tumoral , Femenino , Expresión Génica , Humanos , Inmunidad Celular/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Mucina-1/química , Mucina-1/genética , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Péptidos/administración & dosificación , Péptidos/síntesis química , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Células Plasmáticas/patología , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Conejos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colesterol/metabolismo , Endocitosis/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Membrana Celular/fisiología , Clatrina , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transporte de Proteínas/fisiología , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Animales , Proliferación Celular , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Subgrupos de Linfocitos T/inmunología , Vacunas contra la Tuberculosis/administración & dosificaciónRESUMEN
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.
Asunto(s)
Mitosis , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína smad3/metabolismo , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Femenino , Humanos , Ligandos , Mesodermo/patología , Mitosis/efectos de los fármacos , Neoplasias Ováricas/patología , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
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.