RESUMEN
POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.
Asunto(s)
Ataxia/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , ARN Polimerasa III/genética , Adolescente , Adulto , Ataxia Cerebelosa/genética , Niño , Preescolar , Femenino , Genes Recesivos/genética , Heterocigoto , Humanos , Masculino , Mutación Missense/genética , Proteómica/métodos , Adulto JovenRESUMEN
RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.
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Anodoncia , Anomalías Craneofaciales , Enfermedades Desmielinizantes , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Enfermedades Neurodegenerativas , Humanos , Animales , Ratones , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Mutación/genéticaRESUMEN
The PAQosome (particle for arrangement of quaternary structure) is a 12-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely, the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using the expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119, or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances the binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, the silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modification is a mechanism of client selection.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
INTRODUCTION: The PAQosome is a 12-subunit complex that acts as a co-factor of the molecular chaperones HSP90 and HSP70. This co-chaperone has been shown to participate in assembly and maturation of several protein complexes, including nuclear RNA polymerases, RNA processing factors, the ribosome, PIKKs, and others. Subunits of the PAQosome, adaptors, and clients have been reported to be involved in various diseases, making them interesting targets for drug discovery. AREA COVERED: In this review, the authors cover the detailed mechanisms of PAQosome and chaperone function. Specifically, the authors summarize the status of the PAQosome and some related chaperones and co-chaperones as candidate targets for drug discovery. Indeed, a number of compounds are currently being tested for the development of treatments against diseases, such as cancers and neurodegenerative conditions. EXPERT OPINION: Searching for new drugs targeting the PAQosome requires a better understanding of PAQosome subunit interactions and the discovery of new interaction partners. Thus, PAQosome subunit crystallization is an important experiment to initiate virtual screening against new target and the development of in silico tools such as AlphaFold-multimer could accelerate the search for new interaction partner and determine more rapidly the interaction pocket needed for virtual drug screening.
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Chaperonas Moleculares , Enfermedades Neurodegenerativas , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Unión ProteicaRESUMEN
One of the main burdens in the treatment of diseases is imputable to the delay between the appearance of molecular dysfunctions in the first affected disease cells and their presence in sufficient number for detection in specific tissues or organs. This delay obviously plays in favor of disease progression to an extent that makes efficient treatments difficult, as they arrive too late. The development of a novel medical strategy, termed cell-based interception and precision medicine, seeks to identify dysfunctional cells early, when tissue damages are not apparent and symptoms not yet present, and develop therapies to treat diseases early. Central to this strategy is the use of single-cell technologies that allow detection of molecular changes in cells at the time of phenotypical bifurcation from health to disease. In this article we describe a general procedure to support such an approach applied to neurodegenerative disorders. This procedure combines four components directed towards highly complementary objectives: 1) a high-performance single-cell proteomics (SCP) method (Detect), 2) the development of disease experimental cell models and predictive computational models of cell trajectories (Understand), 3) the discovery of specific targets and personalized therapies (Cure), and 4) the creation of a community of collaborating laboratories to accelerate the development of this novel medical paradigm (Collaborate). A global initiative named 37TrillionCells (37TC) was launched to advance the development of cell-based interception and precision medicine.
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Enfermedades Neurodegenerativas , Humanos , Enfermedades Neurodegenerativas/terapia , Medicina de Precisión/métodos , Atención a la Salud , Proteómica/métodosRESUMEN
Cell-based interception and precision medicine is a novel approach aimed at improving healthcare through the early detection and treatment of diseased cells. Here, we describe our recent progress towards developing cell-based interception and precision medicine to detect, understand, and advance the development of novel therapeutic approaches through a single-cell omics and drug screening platform, as part of a multi-laboratory collaborative effort, for a group of neurodegenerative disorders named leukodystrophies. Our strategy aims at the identification of diseased cells as early as possible to intercept progression of the disease prior to severe clinical impairment and irreversible tissue damage.
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Medicina de Precisión , Humanos , Medicina de Precisión/métodos , Análisis de la Célula Individual/métodos , AnimalesRESUMEN
RNA polymerase III-related leukodystrophy (POLR3-related leukodystrophy) is a rare, genetically determined hypomyelinating disease arising from biallelic pathogenic variants in genes encoding subunits of RNA polymerase III (Pol III). Here, we describe the first reported case of POLR3-related leukodystrophy caused by biallelic pathogenic variants in POLR3D, encoding the RPC4 subunit of Pol III. The individual, a female, demonstrated delays in walking and expressive and receptive language as a child and later cognitively plateaued. Additional neurological features included cerebellar signs (e.g., dysarthria, ataxia, and intention tremor) and dysphagia, while non-neurological features included hypodontia, hypogonadotropic hypogonadism, and dysmorphic facial features. Her MRI was notable for diffuse hypomyelination with myelin preservation of early myelinating structures, characteristic of POLR3-related leukodystrophy. Exome sequencing revealed the biallelic variants in POLR3D, a missense variant (c.541C > T, p.P181S) and an intronic splice site variant (c.656-6G > A, p.?). Functional studies of the patient's fibroblasts demonstrated significantly decreased RNA-level expression of POLR3D, along with reduced expression of other Pol III subunit genes. Notably, Pol III transcription was also shown to be aberrant, with a significant decrease in 7SK RNA and several distinct tRNA genes analyzed. Affinity purification coupled to mass spectrometry of the POLR3D p.P181S variant showed normal assembly of Pol III subunits yet altered interaction of Pol III with the PAQosome chaperone complex, indicating the missense variant is likely to alter complex maturation. This work identifies biallelic pathogenic variants in POLR3D as a novel genetic cause of POLR3-related leukodystrophy, expanding the molecular spectrum associated with this disease, and proposes altered tRNA homeostasis as a factor in the underlying biology of this hypomyelinating disorder.
RESUMEN
The mechanism of assembly of RNA polymerase III (Pol III), the 17-subunit enzyme that synthesizes tRNAs, 5 S rRNA, and other small-nuclear (sn) RNAs in eukaryotes, is not clearly understood. The recent discovery of the HSP90 co-chaperone PAQosome (Particle for Arrangement of Quaternary structure) revealed a function for this machinery in the biogenesis of nuclear RNA polymerases. However, the connection between Pol III subunits and the PAQosome during the assembly process remains unexplored. Here, we report the development of a mass spectrometry-based assay that allows the characterization of Pol III assembly. This assay was used to dissect the stages of Pol III assembly, to start defining the function of the PAQosome in this process, to dissect the assembly defects driven by the leukodystrophy-causative R103H substitution in POLR3B, and to discover that riluzole, an FDA-approved drug for alleviation of ALS symptoms, partly corrects these assembly defects. Together, these results shed new light on the mechanism and regulation of human nuclear Pol III biogenesis.
Asunto(s)
Enfermedades Neurodegenerativas , ARN Polimerasa III , Humanos , ARN Polimerasa III/genética , Riluzol , ARN Polimerasas Dirigidas por ADN , Mutación MissenseRESUMEN
OBJECTIVES: MUC16 (CA125) protein is a high molecular weight mucin overexpressed in the majority of epithelial ovarian cancers (EOC) but not in the epithelium of normal ovaries suggesting that it might play a role in EOC pathogenesis. Here, we explored the phenotypic consequences of MUC16 knockdown and expression of its C-terminal domain with the aim of establishing a role for MUC16 in tumorigenesis. METHODS: MUC16 was down-regulated by stably expressing an anti-MUC16 endoplasmic reticulum-targeted single-chain antibody which prevented MUC16 cell surface localization in NIH:OVCAR3 cells. In addition, we generated epitope tagged, N-terminal region-deleted MUC16 constructs with (MUC16TMU) and without (MUC16CTD) cytoplasmic tail deletions and stably expressed them in SKOV3 cells. RESULTS: Although MUC16 knockdown did not affect the cell growth rate, knockdown cells reached a stationary growth phase after 4 days whereas control cells continued to grow for up to 7 days. Colony formation assays in soft agar demonstrated that MUC16 knockdown cells had >8-fold reduction in their ability to form colonies. Importantly, MUC16 knockdown completely prevents the formation of subcutaneous tumors in nude mice. Conversely, we show that ectopic expression of the MUC16CTD enhances SKOV3 tumor cell growth, colony formation in soft agar and enhances tumor growth and metastases in SCID mice. In addition, MUC16CTD expression increases cell motility, invasiveness, and metastatic property. Deletion of the cytoplasmic tail from the MUC16CTD completely abolished its ability to enhance tumor cell growth, cell motility and invasiveness. Furthermore, the increased invasive properties of MUC16CTD-expressing cells correlated with decreased expression of E-cadherin and increased expression of N-cadherin and vimentin. CONCLUSION: These findings provide the first evidence for a critical role of MUC16 in tumor cell growth, tumorigenesis and metastases.
Asunto(s)
Antígeno Ca-125/biosíntesis , Proteínas de la Membrana/biosíntesis , Animales , Antígeno Ca-125/genética , Cadherinas/biosíntesis , Cadherinas/metabolismo , Carcinoma Epitelial de Ovario , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Transfección , Trasplante Heterólogo , Vimentina/biosíntesisRESUMEN
Introduction: The COVID-19 pandemic originated from the emergence of anovel coronavirus, SARS-CoV-2, which has been intensively studied since its discovery in order to generate the knowledge necessary to accelerate the development of vaccines and antivirals. Of note, many researchers believe there is great potential in systematically identifying host interactors of viral factors already targeted by existing drugs.Areas Covered: Herein, the authors discuss in detail the only available large-scale systematic study of the SARS-CoV-2-host protein-protein interaction network. More specifically, the authors review the literature on two key SARS-CoV-2 drug targets, the Spike surface glycoprotein, and the RNA polymerase. The authors also provide the reader with their expert opinion and future perspectives.Expert opinion: Interactions made by viral proteins with host factors reveal key functions that are likely usurped by the virus and, as aconsequence, points to known drugs that can be repurposed to fight viral infection and collateral damages that can exacerbate various disease conditions in COVID-19.
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Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Animales , COVID-19/virología , Desarrollo de Medicamentos , Reposicionamiento de Medicamentos , Humanos , Mapas de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
BACKGROUND: Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative. RESULTS: CHO cell clones, expressing 300 microg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 microg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential. CONCLUSION: FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.
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Técnicas de Cultivo de Célula/métodos , Medios de Cultivo , Colorantes Fluorescentes , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Separación Celular , Cricetinae , Cricetulus , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Activador de Tejido Plasminógeno/biosíntesisRESUMEN
Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.
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Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Escherichia coli/genética , Péptidos/química , Línea Celular Tumoral , Células Cultivadas , Dimerización , Receptores ErbB/genética , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cuerpos de Inclusión/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Ingeniería de Tejidos , Tirosina/química , Tirosina/metabolismoRESUMEN
Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3aG672E/G672E/Polr3b+/R103Hdouble mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.
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Pérdida del Embrión/enzimología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Mutación/genética , ARN Polimerasa III/genética , Animales , Secuencia de Bases , Pérdida del Embrión/genética , Regulación Enzimológica de la Expresión Génica , Técnicas de Sustitución del Gen , Células HEK293 , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Homocigoto , Humanos , Ratones Endogámicos C57BL , Ratones Mutantes , Actividad Motora , Vaina de Mielina/metabolismo , ARN Polimerasa III/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Delivery of transgenes into specific tissues by adenovirus vectors (AdVs) relies on ablations of their natural tropism and on introduction of a new tropism. If the interaction with its natural receptor is ablated, a new packaging cell line is required to produce the AdV. In the present study, we have used two de novo designed peptides (E-Coil and K-Coil) that interact with each other with high affinity to establish a new receptor-ligand system for the propagation of retargeted AdVs. METHODS: We produced a cell line (293E) expressing on its surface a pseudoreceptor containing the E-Coil. An AdV (AdFK4m/GFP) lacking the interaction with the primary receptor for adenovirus (CAR) and containing the K-Coil inserted at the fiber C-terminus was constructed and tested using two strategies: (1) an RGD motif (Arg-Gly-Asp) was inserted into the HI-loop of the fiber; (2) AdFK4m/GFP was conjugated to a bispecific adaptor for the epidermal growth factor receptor (EGFR). RESULTS: AdFK4m/GFP infected 293E cells more efficiently than cells lacking the pseudoreceptor. The transduction was due to the K-Coil/E-Coil specific interaction since it was competed by addition of soluble K-Coil, but not soluble fiber. We demonstrated that the modified AdV was retargeted toward alpha v integrin by inclusion of the RGD motif, or toward EGFR using the bispecific adaptor. CONCLUSIONS: We have established a new system to produce AdVs ablated of natural tropism. This system should permit the retargeting of AdVs by inserting new ligands within the fiber or through the interaction with bispecific adaptors.
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Adenoviridae/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Péptidos/metabolismo , Internalización del Virus , Replicación Viral , Adenoviridae/genética , Adenoviridae/fisiología , Secuencias de Aminoácidos , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/genética , Humanos , Ligandos , Péptidos/genética , Receptores Virales/metabolismo , Transducción Genética , Ensamble de VirusRESUMEN
BACKGROUND: The receptor for type I insulin like growth factor (IGF-IR) and NFκB signaling both play essential roles in cancer initiation and progression but relatively little is known about possible crosstalk between these pathways. We have shown that the IGF-IR could rescue lung and colon carcinoma cells from Tumor necrosis factor -α (ΤΝF-α)-induced apoptosis by activating autocrine, pro-survival IL-6/gp130/STAT3 signaling, suggesting that IGF-IR expression could alter NF-κB signaling that is required for transcriptional activation of IL-6. OBJECTIVE: Here we sought to determine if and how IGF-IR signaling promotes TNF-α-induced NFκB activation. DESIGN: We used lung carcinoma M-27 and colon carcinoma MC-38 cells to investigate IGF-IR-induced changes to the IKK/IκBα/NFκB pathway by a combination of qPCR, Western blotting, electrophoretic mobility shift assay, a reporter assay and gene silencing. RESULTS: We show that in the presence of increased IGF-IR expression or activation levels, nuclear translocation of NFκB in response to TNF-α was enhanced in lung and colon carcinoma cells and this was due to accelerated phosphorylation and degradation of IκBα. This effect was AKT-dependent and mediated via mitogen-activated protein kinase kinase kinase 3(MEKK3) activation. CONCLUSION: The results suggest that ligand-mediated activation of IGF-IR alters NF-κB signaling in cancer cells in an AKT/MEKK3-dependent manner and that temporal aspects of NF-κB activation can regulate the cytokine profile of the tumor cells and thereby, their interaction with the microenvironment.
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Neoplasias del Colon/metabolismo , Neoplasias Pulmonares/metabolismo , FN-kappa B/metabolismo , Somatomedinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular Tumoral , MAP Quinasa Quinasa Quinasa 3/metabolismo , Ratones , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cross-Talk , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
The IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1. The IGF-Trap has a high affinity for hIGFI and hIGFII but low affinity for insulin, as revealed by surface plasmon resonance. It efficiently blocked IGFIR signaling in several carcinoma cell types and inhibited tumor cell proliferation, migration, and invasion in vitro. In vivo, the IGF-Trap showed favorable pharmacokinetic properties and could suppress the growth of established breast carcinoma tumors when administered therapeutically into tumor-bearing mice, improving disease-free survival. Moreover, IGF-Trap treatment markedly reduced experimental liver metastasis of colon and lung carcinoma cells, increasing tumor cell apoptosis and reducing angiogenesis. Finally, when compared with an anti-IGFIR antibody or IGF-binding protein-1 that were used at similar or higher concentrations, the IGF-Trap showed superior therapeutic efficacy to both inhibitors. Taken together, we have developed a targeted therapeutic molecule with highly potent anticancer effects that could address limitations of current IGFIR-targeting agents.
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Antineoplásicos/farmacología , Carcinoma/metabolismo , Carcinoma/patología , Receptor IGF Tipo 1/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Animales , Especificidad de Anticuerpos , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Células CHO , Carcinoma/tratamiento farmacológico , Carcinoma/mortalidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetulus , Modelos Animales de Enfermedad , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Cinética , Ratones , Metástasis de la Neoplasia , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.