RESUMO
Despite considerable efforts, the mechanisms linking genomic alterations to the transcriptional identity of cancer cells remain elusive. Integrative genomic analysis, using a network-based approach, identified 407 master regulator (MR) proteins responsible for canalizing the genetics of individual samples from 20 cohorts in The Cancer Genome Atlas (TCGA) into 112 transcriptionally distinct tumor subtypes. MR proteins could be further organized into 24 pan-cancer, master regulator block modules (MRBs), each regulating key cancer hallmarks and predictive of patient outcome in multiple cohorts. Of all somatic alterations detected in each individual sample, >50% were predicted to induce aberrant MR activity, yielding insight into mechanisms linking tumor genetics and transcriptional identity and establishing non-oncogene dependencies. Genetic and pharmacological validation assays confirmed the predicted effect of upstream mutations and MR activity on downstream cellular identity and phenotype. Thus, co-analysis of mutational and gene expression profiles identified elusive subtypes and provided testable hypothesis for mechanisms mediating the effect of genetic alterations.
Assuntos
Neoplasias/genética , Transcrição Gênica , Adenocarcinoma/genética , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Células HEK293 , Humanos , Camundongos Nus , Mutação/genética , Reprodutibilidade dos TestesRESUMO
Genome-wide identification of the mechanism of action (MoA) of small-molecule compounds characterizing their targets, effectors, and activity modulators represents a highly relevant yet elusive goal, with critical implications for assessment of compound efficacy and toxicity. Current approaches are labor intensive and mostly limited to elucidating high-affinity binding target proteins. We introduce a regulatory network-based approach that elucidates genome-wide MoA proteins based on the assessment of the global dysregulation of their molecular interactions following compound perturbation. Analysis of cellular perturbation profiles identified established MoA proteins for 70% of the tested compounds and elucidated novel proteins that were experimentally validated. Finally, unknown-MoA compound analysis revealed altretamine, an anticancer drug, as an inhibitor of glutathione peroxidase 4 lipid repair activity, which was experimentally confirmed, thus revealing unexpected similarity to the activity of sulfasalazine. This suggests that regulatory network analysis can provide valuable mechanistic insight into the elucidation of small-molecule MoA and compound similarity.
Assuntos
Algoritmos , Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Antineoplásicos/química , Epistasia Genética , Estudo de Associação Genômica Ampla , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas PequenasRESUMO
Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.
Assuntos
Neoplasias do Colo , Resistencia a Medicamentos Antineoplásicos , Fosfoproteínas , Proteômica , Transdução de Sinais , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteômica/métodos , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/genética , Linhagem Celular Tumoral , Fosforilação , Algoritmos , Proteoma/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies, over the past decade, we developed VESPA-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.
RESUMO
The Mechanism of Action (MoA) of a drug is generally represented as a small, non-tissue-specific repertoire of high-affinity binding targets. Yet, drug activity and polypharmacology are increasingly associated with a broad range of off-target and tissue-specific effector proteins. To address this challenge, we have implemented an efficient integrative experimental and computational framework leveraging the systematic generation and analysis of drug perturbational profiles representing >700 FDA-approved and experimental oncology drugs, in cell lines selected as high-fidelity models of 23 aggressive tumor subtypes. Protein activity-based analyses revealed highly reproducible, drug-mediated modulation of tissue-specific targets, leading to generation of a proteome-wide polypharmacology map, characterization of MoA-related drug clusters and off-target effects, and identification and experimental validation of novel, tissue-specific inhibitors of undruggable oncoproteins. The proposed framework, which is easily extended to elucidating the MoA of novel small-molecule libraries, could help support more systematic and quantitative approaches to precision oncology.
RESUMO
Bi-species, fusion-mediated, somatic cell reprogramming allows precise, organism-specific tracking of unknown lineage drivers. The fusion of Tcf7l1-/- murine embryonic stem cells with EBV-transformed human B cell lymphocytes, leads to the generation of bi-species heterokaryons. Human mRNA transcript profiling at multiple time points permits the tracking of the reprogramming of B cell nuclei to a multipotent state. Interrogation of a human B cell regulatory network with gene expression signatures identifies 8 candidate master regulator proteins. Of these 8 candidates, ectopic expression of BAZ2B, from the bromodomain family, efficiently reprograms hematopoietic committed progenitors into a multipotent state and significantly enhances their long-term clonogenicity, stemness, and engraftment in immunocompromised mice. Unbiased systems biology approaches let us identify the early driving events of human B cell reprogramming.
Assuntos
Reprogramação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Reprogramação Celular/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Feminino , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Multipotentes/metabolismo , Fatores de Transcrição/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/fisiologiaRESUMO
VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused 'click' chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine K(i) and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (K(i)=K(i)(')=1.5+/-0.2microM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, K(i)=0.41+/-0.03microM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, K(i)=1.4+/-0.10microM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.
Assuntos
Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores de beta-Lactamases , beta-Lactamases/metabolismo , Analgésicos/farmacologia , Antibacterianos/farmacologia , Domínio Catalítico , Sinergismo Farmacológico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Mitoxantrona/farmacologia , Modelos Moleculares , Ligação Proteica , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , beta-Lactamases/química , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
Interferon (IFN) gamma's ability to localise in the nucleus and function in gene activation has been known for some time, although the role of the conventional nuclear transporting importin molecules is unclear. Here, we demonstrate for the first time the direct recognition of IFNgamma and an IFNgamma mimetic peptide by IMPalpha and the IMPalpha/beta heterodimer, where the IFNgamma mimetic shows higher affinity. Significantly, this correlates well both with in vivo ability to target green fluorescent protein to the nucleus in transfected cells as determined by quantitative confocal laser scanning microscopy, as well as GAS promoter activity of a luciferase reporter. This has important implications for IFNgamma's anti-viral action, and the potential use of the IFNgamma mimetic in antiviral therapies.
Assuntos
Núcleo Celular/metabolismo , Interferon gama/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Sequência de Aminoácidos , Animais , Materiais Biomiméticos/metabolismo , Células COS , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interferon gama/genética , Dados de Sequência Molecular , Sinais de Localização Nuclear/metabolismo , Peptídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ativação TranscricionalRESUMO
We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.
Assuntos
Antineoplásicos/farmacologia , Tumores Neuroendócrinos/tratamento farmacológico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Estudos de Coortes , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Neoplasias Intestinais/tratamento farmacológico , Neoplasias Intestinais/genética , Tumores Neuroendócrinos/genética , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Medicina de Precisão/métodos , Piridinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genéticaRESUMO
Prostate cancer is the second highest cause of cancer-related deaths of men in the US. Signal transducers and activators of transcription (STATs) proteins are a small family of latent cytoplasmic transcription factors that act downstream of Janus kinase (JAK) activation and mediate intracellular signaling from a wide variety of cytokines, growth factors, and hormones. Aberrant activation of STAT3 has been implicated in the progression of many human carcinomas, including prostate cancer. Previously, we have characterized a novel tyrosine kinase inhibitor peptide, Tkip, that is a mimetic of suppressor of cytokine signaling 1 (SOCS-1). Similar to SOCS-1, Tkip binds to the autophosphorylation site of JAK2 and inhibits phosphorylation of STAT1alpha. In this study, we determined the inhibitory effects of Tkip on the human prostate cancer cell lines DU145 and LNCaP. Tkip inhibited cellular proliferation of both DU145 and LNCaP cells, with a slightly greater antiproliferative effect on DU145 cells. Cell cycle analysis using flow cytometry showed Tkip blockage of progression into the S phase of the cell cycle. Tkip also inhibited constitutive (DU145) and IL-6-induced (LNCaP) activation of STAT3, consistent with the fact that STAT3 activation is mediated by JAK2. Tkip also slightly reduced the levels of cyclin D1, an important regulator of cell cycle progression into S phase, in DU145 and LNCaP cancer cell lines. These data describe a potentially important therapeutic that targets both constitutive and IL-6-induced STAT3 activation in human prostate cancer cell lines.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-6/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Janus Quinase 2 , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Neoplasias da Próstata , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fator de Transcrição STAT3 , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de CitocinaRESUMO
The existence of adult pancreatic progenitor cells has been debated. While some favor the concept of facultative progenitors involved in homeostasis and repair, neither a location nor markers for such cells have been defined. Using genetic lineage tracing, we show that Doublecortin-like kinase-1 (Dclk1) labels a rare population of long-lived, quiescent pancreatic cells. In vitro, Dclk1+ cells proliferate readily and sustain pancreatic organoid growth. In vivo, Dclk1+ cells are necessary for pancreatic regeneration following injury and chronic inflammation. Accordingly, their loss has detrimental effects after cerulein-induced pancreatitis. Expression of mutant Kras in Dclk1+ cells does not affect their quiescence or longevity. However, experimental pancreatitis converts Kras mutant Dclk1+ cells into potent cancer-initiating cells. As a potential effector of Kras, Dclk1 contributes functionally to the pathogenesis of pancreatic cancer. Taken together, these observations indicate that Dclk1 marks quiescent pancreatic progenitors that are candidates for the origin of pancreatic cancer.
Assuntos
Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Pancreatite/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Administração Oral , Animais , Carcinogênese/patologia , Carcinoma Ductal Pancreático/induzido quimicamente , Carcinoma Ductal Pancreático/etiologia , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Quinases Semelhantes a Duplacortina , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/etiologia , Pancreatite/induzido quimicamente , Pancreatite/complicações , Proteínas Serina-Treonina Quinases/genética , Tamoxifeno/administração & dosagemRESUMO
A nuclear localization sequence (NLS) in the type II interferon (IFN) IFN gamma, which is responsible for the nuclear translocation of both the ligand and the alpha-subunit (IFNGR1) of the receptor complex, has previously been characterized and its role in signaling examined in detail. We have now identified an NLS in the type I IFN receptor (IFNAR) common subunit IFNAR1 from humans and show that the human IFNAR1 subunit can translocate to the nucleus following human IFN beta stimulation. An NLS in human IFNAR1 is located in the extracellular domain of IFNAR1 within the sequence (382)RKIIEKKT (numbered for the precursor form). Nuclear import by the NLS functions in a conventional fashion requiring cytosolic import factors, is energy-dependent and inhibited by the prototypical NLS of the SV40 large T-antigen. These studies provide a mechanism for nuclear import of IFNAR1, as well as for type I IFN ligands, and a starting point for studying an alternate role for IFNAR1 in nuclear signaling within the type I IFN system.
Assuntos
Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Sinais de Localização Nuclear , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Transporte Ativo do Núcleo Celular , Trifosfato de Adenosina/metabolismo , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células Clonais , Citosol/metabolismo , Metabolismo Energético , Imunofluorescência , Guanosina Trifosfato/metabolismo , Humanos , Interferon Tipo I/imunologia , Interferon beta/genética , Interferon beta/farmacologia , Interferon gama/química , Interferon gama/genética , Cinética , Ligantes , Proteínas de Membrana , Ficocianina/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TemperaturaAssuntos
Proteínas de Ligação a DNA/química , Animais , DNA/química , DNA de Protozoário/química , DNA Recombinante/química , Polarização de Fluorescência , Glicosídeos/química , Indicadores e Reagentes , Cinética , Leishmania/química , Modelos Moleculares , Oligonucleotídeos/química , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Constitutive phosphoinositide 3-kinase (PI3K)/Akt activation is common in T cell acute lymphoblastic leukemia (T-ALL). Although four distinct class I PI3K isoforms (α, ß, γ, δ) could participate in T-ALL pathogenesis, none has been implicated in this process. We report that in the absence of PTEN phosphatase tumor suppressor function, PI3Kγ or PI3Kδ alone can support leukemogenesis, whereas inactivation of both isoforms suppressed tumor formation. The reliance of PTEN null T-ALL on the combined activities of PI3Kγ/δ was further demonstrated by the ability of a dual inhibitor to reduce disease burden and prolong survival in mice as well as prevent proliferation and promote activation of proapoptotic pathways in human tumors. These results support combined inhibition of PI3Kγ/δ as therapy for T-ALL.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Isoformas de Proteínas , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Classe I de Fosfatidilinositol 3-Quinases , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Desenho de Fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Purinas/química , Purinas/farmacologia , Quinazolinonas/química , Quinazolinonas/farmacologiaRESUMO
We have developed peptide mimetics of gamma interferon (IFN-gamma) that play a direct role in the activation and nuclear translocation of STAT1alpha transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-gamma receptor but rather bind to the cytoplasmic domain of the receptor chain 1, IFNGR-1, and thereby initiate the cellular signaling. Thus, we hypothesized that these mimetics would bypass the poxvirus virulence factor B8R protein that binds to intact IFN-gamma and prevents its interaction with the receptor. Human and murine IFN-gamma mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN-gamma mimetic peptide was also expressed via chemical synthesis with an attached lipophilic group for penetration of cell plasma membrane. In contrast to intact human IFN-gamma, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN-gamma receptor extracellular domain. Expression of B8R protein in WISH cells did not block the antiviral effect of the mimetics against encephalomyocarditis or vesicular stomatitis virus, while the antiviral activity of human IFN-gamma was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN-gamma mimetics was not affected by B8R protein, while IFN-gamma-induced upregulation was blocked. Finally, the mimetics, but not IFN-gamma, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN-gamma can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.
Assuntos
Biomimética , Vírus da Encefalomiocardite/efeitos dos fármacos , Vírus da Encefalomiocardite/metabolismo , Interferon gama/farmacologia , Vaccinia virus/efeitos dos fármacos , Vaccinia virus/metabolismo , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Adenoviridae/genética , Antivirais/farmacologia , Linhagem Celular , Vetores Genéticos , Humanos , Interferon gama/síntese química , Interferon gama/genética , Replicação Viral/efeitos dos fármacosRESUMO
IFN-gamma contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1alpha via a complex of IFN-gamma/IFN-gamma receptor (IFNGR)-1/STAT1alpha with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1alpha. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-gamma resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-gamma and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1alpha, but did not affect the binding of IFN-gamma to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1alpha were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-gamma, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-gamma binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1alpha or the nuclear translocation of IFN-gamma or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-gamma-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-gamma, IFNGR-1, and STAT1alpha.
Assuntos
Interferon gama/metabolismo , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Receptores de Interferon/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Endocitose , Filipina/farmacologia , Humanos , Fator Gênico 3 Estimulado por Interferon , Interferon gama/farmacologia , Fosforilação , Proteínas Recombinantes , Transdução de Sinais , Receptor de Interferon gamaRESUMO
A series of experiments were undertaken to examine the effects of interferon (IFN)-tau on regulation of prostaglandin H synthase (PGHS)-2 mRNA in bovine endometrial (BEND) cells as a means to elucidate the actions of IFN-tau to maintain pregnancy. The objective was to determine if IFN-tau mediates posttranscriptional regulation of PGHS-2 mRNA. Cells were treated with phorbol 12,13-dibutyrate (PdBu) for 3 h to induce PGHS-2 mRNA expression. Actinomycin D (0 or 1 microg/ml) or the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580 (1 microM), were added at 3 h, followed by addition of IFN-tau (0 or 50 ng/ml) at 3.5 h and extraction of RNA at 4.5 h. The concentrations of PGHS-2 mRNA were stable between 3 and 4.5 h regardless of actinomycin D. Simultaneous treatment of PdBu-treated cells with actinomycin D and SB203580 (1 microM) decreased PGHS-2 mRNA. Addition of IFN-tau (50 ng/ml) reduced PGHS-2 mRNA, which was not observed when actinomycin D was present. Concurrent treatments of cells with SB203580 and IFN-tau (5 ng/ml) decreased concentrations of PGHS-2 mRNA in an additive manner. Although IFN-tau reduced PGHS-2 mRNA concentrations, phosphorylation of p38 MAPK was induced by IFN-tau, PdBu, and PdBu combined with IFN-tau after 10 min of treatment. Both the p38 MAPK inhibitor and IFN-tau decreased prostaglandin F(2alpha) secretion, and decreases were additive when the two were given together. In summary, activation of p38 MAPK by PdBu is required for continued presence of PGHS-2 mRNA and secretion of prostaglandin F(2alpha) in BEND cells. Interferon-tau mediates a transcription-dependent mechanism, which induces degradation of PGHS-2 mRNA. However, the consequences of an IFN-tau-induced activation of p38 MAPK warrant further investigation, because inhibition of p38 MAPK caused a degradation of PGHS-2 mRNA.
Assuntos
Endométrio/enzimologia , Interferon Tipo I/farmacologia , Isoenzimas/genética , Proteínas da Gravidez/farmacologia , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/fisiologia , Animais , Bovinos , Ciclo-Oxigenase 2 , Dinoprosta/metabolismo , Sinergismo Farmacológico , Endométrio/citologia , Endométrio/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Imidazóis/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Piridinas/farmacologia , RNA Mensageiro/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Interaction of ligands such as epidermal growth factor and interferon-gamma with the extracellular domains of their plasma membrane receptors results in internalization followed by translocation into the nucleus of the ligand and/or receptor. There has been reluctance, however, to ascribe signaling importance to this, the focus instead being on second messenger pathways, including mobilization of kinases and inducible transcription factors (TFs). The latter, however, fails to explain the fact that so many ligands stimulate the same second messenger cascades/TFs, and yet show distinct gene activation profiles. This is particularly apt in the case of the seven STAT TFs that are held to be the mediators of the distinct cellular functions of over 60 ligands. The current review focuses on five representative nuclear localizing ligands for which there is documentation of translocation into the cytosol and nucleus through well-characterized pathways, in addition to a role in gene activation by ligand/receptor in the nucleus.
Assuntos
Núcleo Celular/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Dimerização , Fator de Crescimento Epidérmico/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Hormônio do Crescimento/metabolismo , Humanos , Interferon gama/metabolismo , Ligantes , Prolactina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Interferon/metabolismo , Transdução de Sinais , Ativação Transcricional , Receptor de Interferon gamaRESUMO
The C-terminus of interferon-gamma (IFNgamma) contains a nuclear localization sequence (NLS) required for the activation and nuclear translocation of the transcription factor STAT1alpha and induction of IFNgamma-activated genes. On the basis of this and other studies, we developed a peptide mimetic of IFNgamma that possesses the IFNgamma functions of antiviral activity and upregulation of MHC class II molecules. The mimetic also shares with IFNgamma the ability to induce the activation and nuclear translocation of STAT1alpha and the IFNgamma receptor (IFNGR)-1 subunit. The mimetic, IFNgamma(95-132), is a peptide that consists of the C-terminal residues 95-132 of murine IFNgamma and contains a required alpha-helical domain and the NLS of IFNgamma. In this study, we determined the mechanism of the intracellular action of the mimetic at the level of signal transduction. We show that the mimetic mediates the nuclear transport of IFNGR-1 through its interaction with IFNGR-1 cytoplasmic region 253-287 via both the helical region and the NLS of IFNgamma(95-132). Alanine substitutions of the NLS of the mimetic showed that the NLS was required for nuclear translocation and that the nuclear transport properties of the mimetic correlated with its ability to bind IFNGR-1. These data also show that the NLS of IFNgamma(95-132) can interact simultaneously with IFNGR-1 and the nuclear import machinery. We found that in in vitro nuclear transport assays tyrosine-phosphorylated STAT1alpha failed to undergo nuclear translocation in the presence of nuclear import factors, but was transported to nucleus in the presence of IFNgamma(95-132) and JAK2-phosphorylated IFNGR-1, to which STAT1alpha binds, as a complex of IFNgamma(95-132)/IFNGR-1/STAT1alpha. Thus, the mimetic, which possesses IFNgamma function, is directly involved as a chaperone in the nuclear transport of STAT1alpha and shares this mechanism of action with that previously described for IFNgamma. The mimetic, like IFNgamma, is able to upregulate the tumor suppressor p21WAF1/CIP1, a direct target of STAT1alpha, and this ability requires the NLS of the mimetic. However, unlike IFNgamma, the mimetic is unable to downregulate c-myc and hence does not inhibit the cycling of cells. This suggests that IFNgamma has additional functions that are not tied directly to the nuclear translocation of STAT1alpha.
Assuntos
Interferon gama/fisiologia , Fragmentos de Peptídeos/fisiologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular/genética , Alanina/genética , Sequência de Aminoácidos , Animais , Ligação Competitiva/genética , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fator Gênico 3 Estimulado por Interferon , Interferon gama/síntese química , Interferon gama/genética , Interferon gama/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Mimetismo Molecular/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Sinais de Localização Nuclear/fisiologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica/genética , Estrutura Secundária de Proteína/genética , Receptores de Interferon/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Receptor de Interferon gamaRESUMO
Superantigens are microbial proteins that induce massive activation, proliferation, and cytokine production by CD4+ T cells via specific Vbeta elements on the TCR. In this study we examine superantigen enhancement of Ag-specific CD4+ T cell activity for humoral B cell responses to T-dependent Ags BSA and HIV gp120 envelope, type I T-independent Ag LPS, and type II T-independent Ag pneumococcal polysaccharides. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B (SEB) 7 days later enhanced the anti-BSA Ab response in mice approximately 4-fold as compared with mice given BSA alone. The anti-gp120 response was enhanced approximately 3-fold by superantigens. The type II T-independent Ag pneumococcal polysaccharide response was enhanced approximately 2.3-fold by superantigens, whereas no effect was observed on the response to the type I T-independent Ag LPS. The superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. SEB-stimulated human CD4+ T cells were examined to determine the role of the mitogen-activated protein (MAP) kinase signal transduction pathway in superantigen activation of T cells. Inhibitors of the mitogen pathway of MAP kinase blocked SEB-induced proliferation and IFN-gamma production, while an inhibitor of the p38 stress pathway had no effect. Consistent with this, SEB activated extracellular signal-regulated kinase/MAP kinase as well as MAP kinase-interacting kinase, a kinase that phosphorylates eIF4E, which is an important component of the eukaryotic protein synthesis initiation complex. Both kinases were inhibited by IL-10. Thus, superantigens enhance humoral immunity via Ag-specific CD4+ T cells involving the stress-independent pathway of MAP kinase.