RESUMO
The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here, we report that, during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes, whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a "seesaw model" in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Estômago/citologiaRESUMO
To allow genome-scale identification of genes that regulate cellular signaling, we cloned >90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase activity-deficient mutants. To establish the utility of this resource, we tested the effect of expression of the kinases on three different cellular signaling models. In all screens, many kinases had a modest but significant effect, apparently due to crosstalk between signaling pathways. However, the strongest effects were found with known regulators and novel components, such as MAP3K10 and DYRK2, which we identified in a mammalian Hedgehog (Hh) signaling screen. DYRK2 directly phosphorylated and induced the proteasome-dependent degradation of the key Hh pathway-regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2 and the known Hh pathway component, GSK3beta. Our results establish kinome expression screening as a highly effective way to identify physiological signaling pathway components and genes involved in pathological signaling crosstalk.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , Fibroblastos/metabolismo , Expressão Gênica , Biblioteca Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Mamíferos , Camundongos , Células NIH 3T3 , Proteínas Oncogênicas/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transativadores/metabolismo , Células Vero , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Quinases DyrkRESUMO
Recognition of viral nucleic acids by pattern recognition receptors initiates type I IFN induction and innate antiviral immune response. Here we show that LSm14A, a member of the LSm family involved in RNA processing in the processing bodies, binds to synthetic or viral RNA and DNA and mediates IRF3 activation and IFN-ß induction. Knockdown of LSm14A inhibits cytosolic RNA- and DNA-trigger type I IFN production and cellular antiviral response. Moreover, LSm14A is essential for early-phase induction of IFN-ß after either RNA or DNA virus infection. We further found that LSm14A-mediated IFN-ß induction requires RIG-I-VISA or MITA after RNA or DNA virus infection, respectively, and viral infection causes translocation of LSm14A to peroxisomes, where RIG-I, VISA, and MITA are located. These findings suggest that LSm14A is a sensor for both viral RNA and DNA and plays an important role in initiating IFN-ß induction in the early phase of viral infection.
Assuntos
Infecções por Vírus de DNA/imunologia , DNA Viral/imunologia , Imunidade Celular/imunologia , Infecções por Vírus de RNA/imunologia , RNA Viral/imunologia , Ribonucleoproteínas/imunologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Proteínas de Membrana/genética , Oligonucleotídeos/genética , Interferência de RNA , Ribonucleoproteínas/genéticaRESUMO
BACKGROUND: An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. RESULTS: By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. CONCLUSION: In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
Assuntos
Anticorpos Monoclonais/química , Biomarcadores Tumorais/imunologia , Proteínas de Ligação a DNA/imunologia , Endonucleases/imunologia , Análise Serial de Proteínas/métodos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Colina-Fosfato Citidililtransferase/imunologia , Colina-Fosfato Citidililtransferase/metabolismo , Reações Cruzadas , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/metabolismoRESUMO
How cellular factors regulate gammaherpesvirus lytic replication is not well understood. Here, through functional screening of a cellular kinase expression library, we identified mitogen-activated protein kinase kinase kinase 8 (MAP3K8/Tpl2) as a positive regulator of murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) lytic gene expression and replication. Tpl2 enhances MHV-68 lytic replication by upregulating lytic gene expression and promoter activities of viral lytic genes, including RTA and open reading frame 57 (ORF57). By screening a cellular transcription factor library, we identified the Fos AP-1 transcription factor as a downstream factor that is both necessary and sufficient for mediating the enhancement of MHV-68 lytic replication by Tpl2. In addition, Tpl2 stimulates the promoter activities of key viral lytic genes, including RTA and ORF57, in an AP-1-dependent manner. We identified an AP-1-responsive element on the MHV-68 RTA promoter as the cis element mediating the upregulation of RTA promoter activity by Tpl2. MHV-68 lytic infection upregulates Fos expression, AP-1 activity, and RTA promoter activity in a Tpl2-dependent manner. We constructed a mutant MHV-68 virus that abolished this AP-1-responsive element. This mutant virus exhibited attenuated lytic replication kinetics, indicative of a critical role of this AP-1-responsive element during lytic replication. Moreover, Tpl2 knockdown inhibited the lytic replication of wild-type MHV-68 (MHV-68-WT) but not that of the MHV-68 mutant virus, indicating that endogenous Tpl2 promotes efficient virus lytic replication through AP-1-dependent upregulation of RTA expression. In summary, through tandem functional screens, we identified the Tpl2/AP-1 signaling transduction pathway as a positive regulator of MHV-68 lytic replication.
Assuntos
MAP Quinase Quinase Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Rhadinovirus/fisiologia , Fator de Transcrição AP-1/fisiologia , Replicação Viral/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Cricetinae , DNA Viral/genética , Genes Virais , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhadinovirus/genética , Transdução de Sinais , Transativadores/genética , Transativadores/fisiologia , Células Vero , Replicação Viral/genéticaRESUMO
Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain.
Assuntos
Trifosfato de Adenosina/farmacologia , Comportamento Animal/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Receptores Purinérgicos P2X/metabolismo , Animais , Cálcio/metabolismo , Potenciais Evocados/efeitos dos fármacos , Feminino , Genoma , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fases de Leitura Aberta/genética , Dor/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X3/deficiência , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X3/metabolismoRESUMO
Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA.
Assuntos
Envelhecimento , Inteligência Artificial , Pesquisa Biomédica , Longevidade , Senescência Celular , Congressos como Assunto , Descoberta de Drogas , Humanos , Estilo de Vida , Preparações FarmacêuticasRESUMO
Fragile X mental retardation protein is an mRNA-binding protein associated with phenotypic manifestations of fragile X syndrome, an X-linked disorder caused by mutation in the FMR1 gene that is the most common inherited cause of intellectual disability. Despite the well-studied genetic mechanism of the disease, the proteoforms of fragile X mental retardation protein have not been thoroughly characterized. Here, we report the expression and mass spectrometric characterization of human fragile X mental retardation protein. FMR1 cDNA clone was transfected into human HEK293 cells to express the full-length human fragile X mental retardation protein. Purified fragile X mental retardation protein was subjected to trypsin digestion and characterized by mass spectrometry. Results show 80.5% protein sequence coverage of fragile X mental retardation protein (Q06787, FMR1_HUMAN) including both the N- and C-terminal peptides, indicating successful expression of the full-length protein. Identified post-translational modifications include N-terminal acetylation, phosphorylation (Ser600), and methylation (Arg290, 471, and 474). In addition to the full-length fragile X mental retardation protein isoform (isoform 6), two endogenous fragile X mental retardation protein alternative splicing isoforms (isoforms 4 and 7), as well as fragile X mental retardation protein interacting proteins, were also identified in the co-purified samples, suggesting the interaction network of the human fragile X mental retardation protein. Quantification was performed at the peptide level, and this information provides important reference for the future development of a targeted assay for quantifying fragile X mental retardation protein in clinical samples. Collectively, this study provides the first comprehensive report of human fragile X mental retardation protein proteoforms and may help advance the mechanistic understanding of fragile X syndrome and related phenotypes associated with the FMR1 mutation.
RESUMO
Programmed cell death 1 (PD-1)/PD-1 ligand-1 (PD-L1)-blocking monoclonal antibodies (mAbs) have taken center stage for tumor immune checkpoint therapy. Identification of the "hotspots" on PD-1 for mAbs will help to develop next-generation oral deliverable agents with long-lasting efficacy. Here, we identified two PD-1-targeting mAbs, GY-5 and GY-14, with PD-1/PD-L1-blocking efficacy. Complex structural information revealed that both mAbs mainly bind to the FG loop of PD-1, which also contributes multiple interactions with PD-L1. The FG loop adopts substantially varied conformations upon binding to different mAbs, providing a novel targetable region for the development of PD-1-specific biologics and small chemical molecules. Glycosylation modifications of PD-1 could be observed in three of the four potential N-linked glycosylation sites. However, the binding of GY-5 and GY-14 to PD-1 was not affected by glycosylation. These findings broaden our understanding of the mechanism of anti-PD-1 mAbs and provide insight into the development of agents targeting PD-1.
RESUMO
Purpose: The TP53 tumor-suppressor gene is mutated in >95% of high-grade serous ovarian cancers. Detecting an autologous antibody response to TP53 that might improve early detection.Experimental Design: An immunoassay was developed to measure TP53 autoantibody in sera from 378 cases of invasive epithelial ovarian cancer and 944 age-matched healthy controls from the United States, Australia, and the United Kingdom. Serial preclinical samples from cases and controls were also assayed from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).Results: Using a cutoff value of 78 U/mL to achieve a specificity of 97.4%, TP53 autoantibody was elevated in 30% of 50 cases from MD Anderson, 21.3% of 108 cases from the Australian Ovarian Cancer Study, and 21% of 220 cases from the UKCTOCS. Among 164 cases with rising CA125 detected with the UKCTOCS risk of ovarian cancer algorithm (ROCA), 20.7% had elevated TP53 autoantibody. In cases missed by the ROCA, 16% of cases had elevated TP53 autoantibody. Of the 34 ovarian cancer cases detected with the ROCA, TP53 autoantibody titers were elevated 11.0 months before CA125. In the 9 cases missed by the ROCA, TP53 autoantibody was elevated 22.9 months before cancer diagnosis. Similar sensitivity was obtained using assays with specific mutant and wild-type TP53.Conclusions: TP53 autoantibody levels provide a biomarker with clinically significant lead time over elevation of CA125 or an elevated ROCA value. Quantitative assessment of autoantibodies in combination with CA125 holds promise for earlier detection of invasive epithelial ovarian cancer. Clin Cancer Res; 23(19); 5912-22. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Proteínas de Membrana/sangue , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Ovarianas/sangue , Proteína Supressora de Tumor p53/sangue , Adulto , Idoso , Austrália , Autoanticorpos/sangue , Carcinoma Epitelial do Ovário , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Proteína Supressora de Tumor p53/imunologia , Reino Unido , Estados UnidosRESUMO
Glial-derived nexin (GDN) is a proteinase inhibitor secreted from glial cells and it can enhance neuronal function. However, its expression and function in neuronal differentiation are not, as yet, well-known. In the present study, we analyzed glial-derived nexin gene expression in dissociated neural stem/progenitor cells (NS/PCs) (D0) from the embryonic mouse cerebral cortex, expanded NS/PC cultures (D4 and D10 cultures) and cultured neurons (E15) using a semi-quantitative RT-PCR assay. Our data suggest that mouse GDN, homologue of human GDN, was significantly up-regulated in the expanded NS/PC cultures and cultured neurons. To analyze its function in neuronal differentiation, human GDN cDNA was cloned into bicistronic plasmids containing green fluorescent protein (GFP) and the resulting plasmids were transfected into rodent primary NS/PCs and non-neuronal human embryonic kidney (HEK) cells. Our data suggest that the ectopic expression of human GDN triggered the expression of the neuronal marker TuJ1 in both NS/PCs and HEK cells. We conclude that GDN is up-regulated during neuronal differentiation and plays a role in transforming non-neuronal HEK cells into neuron-like cells.
Assuntos
Precursor de Proteína beta-Amiloide/fisiologia , Diferenciação Celular/fisiologia , Expressão Gênica/fisiologia , Neurônios/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Northern Blotting/métodos , Bromodesoxiuridina/metabolismo , Células Cultivadas , Clonagem Molecular/métodos , Embrião de Mamíferos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica/métodos , Indóis , Camundongos , Neurônios/citologia , Nexinas de Proteases , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células-Tronco/fisiologia , Transfecção/métodos , Tubulina (Proteína)/metabolismoRESUMO
Three cell groups, neural stem/progenitor cells (NS/PCs) dissociated from the embryonic day 11 (E11) rodent cerebral cortex, expanded NS/PC cultures, and cultured neurons from E15, were used to conduct a genomic study with differential display (DD). The mouse Af1q, homologue of human AF1q, was found to be significantly up-regulated during the neuronal production from NS/PCs. The ectopic expression of human AF1q triggered the expression of the neuronal marker TuJ1 in non-neuronal human embryonic kidney (HEK) cells.
Assuntos
Proteínas Sanguíneas/genética , Rim/citologia , Proteínas de Neoplasias/genética , Neurônios/citologia , Células-Tronco/citologia , Animais , Biomarcadores , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Neurônios/fisiologia , Proteínas Proto-Oncogênicas , Células-Tronco/fisiologia , Transfecção , Tubulina (Proteína)/genéticaRESUMO
Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin. By including protease cleavage sites between GLUC and beta-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.