RESUMO
Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/ultraestrutura , Microscopia de Força Atômica/métodos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Água/química , Receptores de Inositol 1,4,5-Trifosfato , Conformação Proteica , SoluçõesRESUMO
Infections of bacteria and viruses induce host defense reactions known as innate responses that include the production of cytokines and chemokines. The production of type I interferon (IFN) is known to be induced by viral double-stranded (ds) RNA or bacterial lipopolysaccharide (LPS). Although important functions for the transcription factors NF-kappaB and interferon regulatory factor-3 (IRF-3) are indicated, the molecular signals leading to the activation of IFN genes have yet to be elucidated. We provide several lines of evidence that LPS and dsRNA trigger distinct intracellular signals upstream. Notably, our investigation revealed a critical function for TIRAP/MAL, a signaling adapter for Toll-like receptor (TLR) 4, in LPS-induced but not dsRNA-induced activation of IRF-3. These results highlight cross-talk between TLR-mediated and virus/dsRNA-induced signals resulting in activation of the IFN system.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana , Receptores de Interleucina-1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Humanos , Fator Regulador 3 de Interferon , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA de Cadeia Dupla/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Interferon (IFN) regulatory factor-3 (IRF-3) is a unique member of the IRF family. Its transcriptional activity is regulated solely by posttranslational modifications. We review current knowledge of the mechanism of IRF-3 activation: signalling triggered by infections including viruses and bacteria, phosphorylation of IRF-3 on certain serine residues, homodimer formation, and active holocomplex formation with coactivator CBP/p300.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Previsões , Humanos , Fator Regulador 3 de Interferon , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , RNA de Cadeia Dupla/farmacologia , Transdução de Sinais , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional , Viroses/metabolismoRESUMO
There is accumulating evidence demonstrating that HIF-1 functions as a key regulator of the adaptation responses to hypoxia in cancer tissues. To this evidence, we add that adaptation responses to glucose deprivation plus hypoxia are also necessary for the survival of tumor cells in the tumor microenvironment as cancer tissues are exposed to glucose deprivation as well as hypoxia. We found that adrenomedullin (AM), VEGF, Glut-1, Glut-3, and Hexokinase-2 among 45 hypoxia-inducible genes investigated were expressed at higher levels under glucose-deprived hypoxic conditions than under hypoxic conditions. Glucose deprivation activated the AMPK under normoxia and hypoxia. Compound C, an inhibitor of AMPK, suppressed the expressions of AM and VEGF which had already been enhanced under glucose-deprived hypoxic conditions. siRNAs for both AMPKalpha1 and AMPKalpha2 suppressed the expressions of AM and VEGF. HIF-1alpha protein level and the transcriptional activity of HIF-1 under glucose-deprived hypoxic conditions were thus found to be similar to those under hypoxic conditions. Furthermore, tumor cells in 15 out of 20 human pancreatic cancer tissue specimens were stained by anti-phospho-AMPKalpha antibody. Our results thus suggest that the enhanced expressions of those genes mediated by the activation of AMPK and HIF-1 therefore play a pivotal role in the tumor formation of pancreatic cancers.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/biossíntese , Glucose/fisiologia , Hexoquinase/biossíntese , Fator 1 Induzível por Hipóxia/biossíntese , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Quinases/biossíntese , Quinases Proteína-Quinases Ativadas por AMP , Animais , Biomarcadores Tumorais/biossíntese , Hipóxia Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Hipóxia , Camundongos , Camundongos SCID , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/irrigação sanguínea , Fosforilação , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
Infections of bacteria and viruses induce host defense reactions known as innate responses including the activation of interferon regulatory factor-3 (IRF-3), critical for the activation of type I interferon system. Upon immediate early signals triggered by the infection, IRF-3 is phosphorylated and a homodimer results. The homodimer complexes with the coactivator CREB-binding protein (CBP)/p300 in the nucleus; thus, holocomplex of IRF-3 competent in DNA binding is generated. We showed CBP/p300 to be indispensable for the DNA binding activity of the holocomplex and to aid the binding through direct interaction with the DNA. We demonstrated that p300 binds with the IRF-3 homodimer via a Q-rich domain and that an intact histone acetyltransferase (HAT) domain is indispensable for the DNA binding of the holocomplex along with a CH3 domain, which connects the HAT and Q-rich domains. These results highlight a novel function of CBP/p300: direct involvement in sequence-specific DNA binding. Furthermore, the critical function of these domains in virus-induced gene activation was demonstrated in vivo by using p300 mutants.
Assuntos
Acetiltransferases/química , Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Acetiltransferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Ácido Desoxicólico/farmacologia , Dimerização , Glutationa Transferase/metabolismo , Glutationa Transferase/farmacologia , Glicerol/farmacologia , Células HeLa , Histona Acetiltransferases , Humanos , Immunoblotting , Fator Regulador 3 de Interferon , Interferon Tipo I/metabolismo , Camundongos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/farmacologia , Transfecção , Raios Ultravioleta , Fatores de Transcrição de p300-CBPRESUMO
Transcription factor IRF-3 is post-translationally activated by Toll-like receptor (TLR) signaling and has critical roles in the regulation of innate immunity. Here we present the X-ray crystal structure of the C-terminal regulatory domain of IRF-3(175-427) (IRF-3 175C) at a resolution of 2.3 A. IRF-3 175C is structurally similar to the Mad homology domain 2 of the Smad family. Structural and functional analyses reveal phosphorylation-induced IRF-3 dimerization, which generates an extensive acidic pocket responsible for binding with p300/CBP. Although TLR and Smad signaling are evolutionarily independent, our results suggest that IRF-3 originates from Smad and acquires its function downstream of TLR.