RESUMO
BACKGROUND: Ovarian cancer stem cells (OCSCs) are the main cause of relapse and drug resistance in patients with ovarian cancer. Anisomycin has been shown to be an effective antitumor agent, but its mechanism of action in ovarian cancer remains elusive. METHODS: CD44+/CD133+ human OCSCs were isolated from human ovarian cancer tissues. OCSCs were interfered with using anisomycin and specific small-interfering RNA (siRNA). Microarray assay, MTT, in vivo tumorigenic experiments, transwell assay, cell cycle assay, colony formation assay, angiogenesis assay, and hematoxylin and eosin staining were used to detect the mechanism of anisomycin with respect to inhibiting the activity of OCSCs. Expression of the NCBP2-AS2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) pathway was examined using western blotting, a quantitative real-time PCR (RT-qPCR) and immunofluorescence staining. Bioinformatics analysis was used for predictive analysis of NCBP2-AS2 expression in urogenital tumors. RESULTS: Microarray analysis showed that treatment with anisomycin significantly decreased the expression of antisense RNA NCBP2-AS2 in OCSCs. In vitro cellular experiments showed that interfering with endogenous antisense RNA NCBP2-AS2 using siRNA distinctly inhibited the proliferation, migration and angiogenesis of OCSCs, whereas in vivo animal experiments revealed decreased tumorigenesis in nude mice. Moreover, the results of RT-qPCR and western blotting demonstrated that both anisomycin treatment and NCBP2-AS2 silencing led to significant reductions in the mRNA and protein expression levels of NCBP2-AS2, MEK, ERK and STAT3. From a bioinformatic point of view, antisense RNA NCBP2-AS2 exhibited significantly differential expression between urogenital tumors and normal controls, and a similar expression pattern was found in the genes NCBP2, RPL35A, DNAJC19 and ECE2, which have similarity to NCBP2-AS2. CONCLUSIONS: Anisomycin suppresses the in vivo and in vitro activity of human OCSCs by downregulating the antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling pathway, whereas the antisense RNA NCBP2-AS2 and genes with similarity have the potential to serve as markers for clinical diagnosis and prognosis of urogenital tumors.
Assuntos
Neoplasias Ovarianas , Animais , Camundongos , Humanos , Feminino , Anisomicina/metabolismo , Anisomicina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fator de Transcrição STAT3/genética , Camundongos Nus , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Transdução de Sinais , RNA Interferente Pequeno/uso terapêutico , Células-Tronco Neoplásicas/metabolismo , Proliferação de Células/genéticaRESUMO
Intervertebral disc degeneration (IVDD) mainly manifests as an imbalance between the synthesis and degradation of cellular and extracellular matrix (ECM) components. The cytokine interleukin (IL)-1ß-induced inflammatory response of intervertebral discs causes ECM degradation. The aim of this study was to investigate the effects of a 970-nm diode laser therapy (DLT) on inflammatory cytokine IL-1ß and ECM degradation proteinases in nucleus pulposus (NP) tissues in a puncture-induced rabbit IVDD model. Thirty-six New Zealand white rabbits were randomly divided into six groups: the normal group, IVDD group, laser group, sham laser group, IVDD + anisomycin (p38MAPK signaling pathway agonist), and laser + anisomycin group. Effects of laser on IVDD progression were detected using radiographic and magnetic resonance imaging. Hematoxylin and eosin, Alcian blue, safranin O-fast green staining, western blotting, and immunohistochemistry staining were performed for the histological analysis and molecular mechanism underlying protection against puncture-induced matrix degradation in NP tissues by DLT. DLT reduced the degree of disc degeneration in the gross anatomy of the disc and increased the T2-weighted signal intensity of NP. Inflammatory cytokine IL-1ß levels in the disc were significantly reduced after DLT suppressed the matrix-degrading proteinases MMP13 and ADAMTS-5 and upregulated the protein expression of collagen II and aggrecan. Moreover, it inhibited the p38MAPK signaling pathway in NP tissues in a puncture-induced rabbit IVDD model. DLT reduced puncture-induced overexpression of inflammatory cytokines, mainly IL-1ß, thus inhibiting matrix degeneration of NP tissues and ameliorating IVDD. This may be related to inhibition of the p38 MAPK signaling pathway.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Coelhos , Animais , Degeneração do Disco Intervertebral/radioterapia , Lasers Semicondutores/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anisomicina/metabolismo , Citocinas/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Morroniside can prevent myocardial injury caused by ischemia and hypoxia, which can be used to treat acute myocardial infarction (AMI). Hypoxia can cause apoptosis and autophagic death of cardiomyocytes. Morroniside has the ability to inhibit apoptosis and autophagy. However, the relationship between Morroniside-protected cardiomyocytes and two forms of death is unclear. The effects of Morroniside on the proliferation, apoptosis level, and autophagic activity of rat cardiomyocyte line H9c2 under hypoxia were first observed. Next, the roles of Morroniside in the phosphorylation of JNK and BCL2, BCL2-Beclin1, and BCL2-Bax complexes as well as mitochondrial membrane potential in H9c2 cells were evaluated upon hypoxia. Finally, the significance of BCL2 or JNK in Morroniside-regulated autophagy, apoptosis, and proliferation in H9c2 cells was assessed by combining Morroniside and BCL2 competitive inhibitor (ABT-737) or JNK activator (Anisomycin). Our results showed that hypoxia promoted autophagy and apoptosis of H9c2 cells, and inhibited their proliferation. However, Morroniside could block the effect of hypoxia on H9c2 cells. In addition, Morroniside could inhibit JNK phosphorylation, BCL2 phosphorylation at the Ser70 and Ser87 sites, and the dissociation of BCL2-Beclin1 and BCL2-Bax complexes in H9c2 cells upon hypoxia. Moreover, the reduction of mitochondrial membrane potential in H9c2 cells caused by hypoxia was improved by Morroniside administration. Importantly, the inhibited autophagy, apoptosis, and promoted proliferation in H9c2 cells by Morroniside were reversed by the application of ABT-737 or Anisomycin. Overall, Morroniside inhibits Beclin1-dependent autophagic death and Bax-dependent apoptosis via JNK-mediated BCL2 phosphorylation, thereby improving the survival of cardiomyocytes under hypoxia.
Assuntos
Apoptose , Miócitos Cardíacos , Ratos , Animais , Miócitos Cardíacos/metabolismo , Proteína Beclina-1 , Proteína X Associada a bcl-2/metabolismo , Fosforilação , Anisomicina/metabolismo , Anisomicina/farmacologia , Autofagia , Hipóxia/metabolismoRESUMO
AIMS: Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms. METHODS: ALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies. RESULTS: AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing. CONCLUSIONS: In summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.
Assuntos
Falência Hepática Aguda , Sistema de Sinalização das MAP Quinases , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Acetaminofen/metabolismo , Lipopolissacarídeos/farmacologia , Anisomicina/metabolismo , Anisomicina/farmacologia , Fígado , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/prevenção & controle , Piridonas/farmacologia , Necrose/metabolismo , Camundongos Endogâmicos C57BL , HepatócitosRESUMO
INTRODUCTION: Social buffering is the phenomenon, in which stress and fear reactions caused by exposure to stressful stimuli when animals are exposed to homogeneous relationships are attenuated. Social buffering reduces fear memory behavior such as escape, avoidance, and freezing behavior in rodents due to social existence. Here, we aimed to determine alterations of fear behavior and neural activity in the medial prefrontal cortex (mPFC) in response to the presence of another rat in fear-exposed conditions and to confirm the role of oxytocin in mPFC in regulating social buffering. METHODS: We performed a passive avoidance test and determined positive c-Fos expression in single- and pair-exposed rats. Anisomycin (a protein synthesis inhibitor) and oxytocin receptor regulators (carbetocin; agonist and atosiban; antagonist) were microinjected into the mPFC to clarify the role of oxytocin in the mPFC. RESULTS: While single-exposed rats showed a significant increase in both freezing and passive avoidance behaviors compared to control rats, pair-exposed rats showed significantly less fear behavior compared to single-exposed rats. The c-Fos expression in the prelimbic (PL) mPFC was significantly increased in pair-exposed rats compared to that in control and single-exposed rats. The pair-exposed effect was blocked by anisomycin injections into the PL mPFC of pair-exposed rats. Furthermore, when a carbetocin was injected into the PL mPFC in single-exposed rats, fear behavior was decreased, and these changes were blocked by atosiban. DISCUSSION: Our findings suggest that reduction of fear-related behavior induced by acute pair-exposure is mediated by oxytocin receptors in the PL mPFC. Pair exposure with conspecifics during fear-inducing situations helps coping with fear by significantly increasing the role of oxytocin in the PL mPFC.
Assuntos
Ocitocina , Córtex Pré-Frontal , Animais , Anisomicina/metabolismo , Anisomicina/farmacologia , Medo/fisiologia , Ocitocina/metabolismo , Ocitocina/farmacologia , Córtex Pré-Frontal/metabolismo , Ratos , Receptores de Ocitocina/metabolismoRESUMO
Long-term potentiation (LTP) is generally divided into two phases, early (E-) and late (L-) LTP, of which only L-LTP is thought to depend on protein synthesis. Here we report that E-LTP can also be dependent on protein synthesis at higher levels of synaptic activation. Moreover, we show that the requirement for protein synthesis during L-LTP extends beyond the early induction phase and that it depends on synaptic stimulation. This suggests that the level of neuronal activity is a crucial determinant for the role of protein synthesis in E- and L-LTP.
Assuntos
Potenciação de Longa Duração/fisiologia , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia , Sinapses/metabolismo , Animais , Anisomicina/metabolismo , Eletrofisiologia , Neurônios/citologia , Inibidores da Síntese de Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão SinápticaRESUMO
The peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor family and plays a central role in the regulation of lipid metabolism, glucose homeostasis and inflammatory processes. In addition to its ligand-induced activation, PPARalpha is regulated by phosphorylation via ERK-MAPK, PKA and PKC. In this study we examined the effect of p38-MAPK on PPARalpha transcriptional activity. In COS-7 cells, anisomycin, a p38 activator, induced a dose-dependent phosphorylation of PPARalpha and a 50% inhibition of its transcriptional activity. In H4IIE hepatoma cells, anisomycin-induced p38 phosphorylation decreased both endogenous and PPARalpha ligand-enhanced L-CPTI and ACO gene expression. Interestingly, PPARalpha/p38 interaction required the molecular adapter ZIP/p62. Reducing ZIP/p62 expression by siRNA, partially reversed the inhibitory effect of anisomycin on L-CPTI gene expression. In conclusion, we showed that p38 activation induced PPARalpha phosphorylation and inhibition of its transcriptional activity through a trimeric interaction between p38-MAPK, ZIP/p62 and PPARalpha.
Assuntos
Proteínas de Choque Térmico/metabolismo , PPAR alfa/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anisomicina/metabolismo , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Inibidores da Síntese de Ácido Nucleico/metabolismo , PPAR alfa/genética , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteína Sequestossoma-1 , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Keratins are crucial for the anchorage of desmosomes. Severe alterations of keratin organization and detachment of filaments from the desmosomal plaque occur in the autoimmune dermatoses pemphigus vulgaris and pemphigus foliaceus (PF), which are mainly caused by autoantibodies against desmoglein (Dsg) 1 and 3. Keratin alterations are a structural hallmark in pemphigus pathogenesis and correlate with loss of intercellular adhesion. However, the significance for autoantibody-induced loss of intercellular adhesion is largely unknown. In wild-type (wt) murine keratinocytes, pemphigus autoantibodies induced keratin filament retraction. Under the same conditions, we used murine keratinocytes lacking all keratin filaments (KtyII k.o.) as a model system to dissect the role of keratins in pemphigus. KtyII k.o. cells show compromised intercellular adhesion without antibody (Ab) treatment, which was not impaired further by pathogenic pemphigus autoantibodies. Nevertheless, direct activation of p38MAPK via anisomycin further decreased intercellular adhesion indicating that cell cohesion was not completely abrogated in the absence of keratins. Direct inhibition of Dsg3, but not of Dsg1, interaction via pathogenic autoantibodies as revealed by atomic force microscopy was detectable in both cell lines demonstrating that keratins are not required for this phenomenon. However, PF-IgG shifted Dsg1-binding events from cell borders toward the free cell surface in wt cells. This led to a distribution pattern of Dsg1-binding events similar to KtyII k.o. cells under resting conditions. In keratin-deficient keratinocytes, PF-IgG impaired Dsg1-binding strength, which was not different from wt cells under resting conditions. In addition, pathogenic autoantibodies were capable of activating p38MAPK in both KtyII wt and k.o. cells, the latter of which already displayed robust p38MAPK activation under resting conditions. Since inhibition of p38MAPK blocked autoantibody-induced loss of intercellular adhesion in wt cells and restored baseline cell cohesion in keratin-deficient cells, we conclude that p38MAPK signaling is (i) critical for regulation of cell adhesion, (ii) regulated by keratins, and (iii) targets both keratin-dependent and -independent mechanisms.
Assuntos
Adesão Celular/genética , Desmogleínas/metabolismo , Queratinócitos/fisiologia , Queratinas/metabolismo , Pênfigo/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anisomicina/metabolismo , Autoanticorpos/metabolismo , Células Cultivadas , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Pênfigo/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
Histone deacetylase 1 (HDAC1) is a major regulator of chromatin structure and gene expression. Tight control of HDAC1 expression is essential for normal cell cycle progression of mammalian cells. HDAC1 mRNA levels are regulated by growth factors and by changes in intracellular deacetylase activity levels. Stimulation of the mitogen-activated protein kinase cascade by anisomycin or growth factors, together with inhibition of deacetylases by trichostatin A (TSA), leads to stable histone H3 phosphoacetylation and strongly induced HDAC1 expression. In contrast, activation of the nucleosomal response by anisomycin alone results only in transient phosphoacetylation of histone H3 without affecting HDAC1 mRNA levels. The transcriptional induction of the HDAC1 gene by anisomycin and TSA is efficiently blocked by H89, an inhibitor of the nucleosomal response. Detailed studies of the kinetics of histone acetylation and phosphorylation show that the two modifications are synergistic and essential for induced HDAC1 transcription. Activation of the HDAC1 gene by anisomycin together with TSA or by growth factors is accompanied by phosphoacetylation of HDAC1 promoter-associated histone H3. Our results present evidence for a precise regulatory mechanism which allows induction of the HDAC1 gene in response to proliferation signals and modulation of HDAC1 expression dependent on intracellular deacetylase levels.
Assuntos
Regulação da Expressão Gênica , Histona Desacetilases/genética , Histonas/metabolismo , Células 3T3 , Acetilação , Animais , Anisomicina/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Genes Precoces , Substâncias de Crescimento/metabolismo , Histona Desacetilase 1 , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Nucleossomos/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/metabolismo , RNA Mensageiro/metabolismoRESUMO
Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Endorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Conformação de Ácido Nucleico , Peptidil Transferases/antagonistas & inibidores , Inibidores da Síntese de Proteínas/metabolismo , RNA Ribossômico 28S/metabolismo , Ribossomos/metabolismo , Ricina/metabolismo , Animais , Anisomicina/metabolismo , Antibacterianos/metabolismo , Sequência de Bases , Sítios de Ligação , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , Dados de Sequência Molecular , Nucleosídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Ribossômico 28S/química , Ratos , Transdução de SinaisRESUMO
Ultraviolet (UV) radiation is a carcinogen that generates DNA lesions. Here, we demonstrate an unexpected role for DGCR8, an RNA binding protein that canonically functions with Drosha to mediate microRNA processing, in the repair of UV-induced DNA lesions. Treatment with UV induced phosphorylation on serine 153 (S153) of DGCR8 in both human and murine cells. S153 phosphorylation was critical for cellular resistance to UV, the removal of UV-induced DNA lesions, and the recovery of RNA synthesis after UV exposure but not for microRNA expression. The RNA-binding and Drosha-binding activities of DGCR8 were not critical for UV resistance. DGCR8 depletion was epistatic to defects in XPA, CSA, and CSB for UV sensitivity. DGCR8 physically interacted with CSB and RNA polymerase II. JNKs were involved in the UV-induced S153 phosphorylation. These findings suggest that UV-induced S153 phosphorylation mediates transcription-coupled nucleotide excision repair of UV-induced DNA lesions in a manner independent of microRNA processing.
Assuntos
Dano ao DNA , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Animais , Anisomicina/metabolismo , Antracenos/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Reparo do DNA , Células HCT116 , Células HeLa , Humanos , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Camundongos , Fosforilação , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Raios UltravioletaRESUMO
The binding of radiolabeled T-2 to eukaryotic ribosomes was studied. The toxin bound to ribosomes in a time-, temperature- and concentration-dependent manner. The binding was saturable (0.3 nM), reversible at 37 degrees (half-time approximately 2.5 hr) and specific. The stoichiometry was one toxin molecule bound per ribosome. Binding of T-2 appeared to stablize the toxin recognition site to thermal degradation. A synthetically derived epimer of T-2 bound to the same ribosomal site as authentic T-2, but apparently with lower affinity. Two other trichothecene toxins tested blocked the binding of T-2 to ribosomes in a manner reflecting their protein synthesis inhibitory potencies. Anisomycin blocked the binding of T-2 to both isolated ribosomes and cells, whereas emetine blocked binding only to cells. Our data, together with that in the accompanying paper (Middlebrook JL and Leatherman DL, Biochem Pharmacol 38: 3093-3102, 1989), suggest that T-2 interaction with CHO cells is best viewed as a free, bidirectional movement of toxin across the plasma membrane and specific high-affinity binding to ribosomes.
Assuntos
Ribossomos/metabolismo , Sesquiterpenos/metabolismo , Toxina T-2/metabolismo , Animais , Anisomicina/metabolismo , Ligação Competitiva , Linhagem Celular , Cricetinae , Cinética , Biossíntese de Proteínas , Estereoisomerismo , TemperaturaAssuntos
Biossíntese de Proteínas , Ribossomos/metabolismo , Anisomicina/metabolismo , Azidas/farmacologia , Sítios de Ligação , Cicloeximida/farmacologia , Toxina Diftérica/farmacologia , Doxiciclina/farmacologia , Ácido Fusídico/farmacologia , Modelos Biológicos , Elongação Traducional da Cadeia Peptídica , Polirribossomos/metabolismo , Conformação Proteica , Puromicina/metabolismo , Proteínas Ribossômicas , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-AtividadeAssuntos
Alcaloides/farmacologia , Fenantridinas/farmacologia , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Alcaloides de Amaryllidaceae , Anisomicina/metabolismo , Resistência Microbiana a Medicamentos , Proteínas Fúngicas/biossíntese , Cinética , Mutação , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Alongamento de Peptídeos , Fenilalanina/metabolismo , Plantas , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Puromicina/metabolismo , Receptores de Droga , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Especificidade da Espécie , Fatores de TempoRESUMO
Eleven mutations that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit, have been identified in 23S rRNA. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by the binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions and (2) those that stabilize the apo conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances and are reversed when A-site substrates bind to the ribosome.
Assuntos
Anisomicina/metabolismo , Resistência Microbiana a Medicamentos/genética , Haloarcula marismortui/metabolismo , Mutação , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/metabolismo , Ribossomos , Anisomicina/química , Sítios de Ligação , Modelos Moleculares , Estrutura Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Inibidores da Síntese de Proteínas/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismoRESUMO
OBJECTIVE: JNK regulates matrix metalloproteinase (MMP) gene expression and joint destruction in rheumatoid arthritis (RA). Previous studies demonstrated that the 2 upstream MAPK kinases (MKK-4 and MKK-7) are phosphorylated in RA synovium and form a complex with JNK in fibroblast-like synoviocytes (FLS). However, the functional hierarchy of MKK-4 and MKK-7 in FLS has not been determined. We determined the relative contributions of these MKKs by evaluating the effect of MKK-4 and MKK-7 gene knockdown in cultured FLS. METHODS: FLS were transfected with MKK-4 and/or MKK-7 small interfering RNA, and protein levels were determined by immunoblotting. After stimulation with interleukin-1/beta (IL-1beta), tumor necrosis factor alpha(TNFalpha, or anisomycin, kinase function was determined by in vitro kinase assay. Activator protein 1 (AP-1) binding and transcriptional activity were determined by electrophoretic mobility shift assay and AP-1-luciferase promoter assay, respectively. MMP-3 expression was determined by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: IL-1beta-induced JNK phosphorylation was dependent on MKK-7 but not on MKK-4; however, anisomycin-activated JNK required both kinases. In vitro kinase assay demonstrated that IL-1beta-or TNFalpha induced JNK activity was only MKK-7 dependent, while anisomycin-activated JNK was both MKK-4 and MKK-7 dependent. IL-1beta-induced AP-1 binding activity and AP-1-driven gene expression were strictly MKK-7 dependent. Finally, MMP-3 production only required MKK-7, and there was no effect of MKK-4 deficiency. CONCLUSION: These data indicate that only MKK-7 is required for JNK activation in FLS after cytokine stimulation; however, other forms of cellular stress utilize MKK-4. Thus, JNK function might be modulated by targeting MKK-7 to suppress cytokine-mediated FLS activation while leaving other stress responses intact.
Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase 7/metabolismo , Membrana Sinovial/metabolismo , Anisomicina/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/fisiopatologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Interleucina-1/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase 7/genética , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/fisiopatologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
1. Of the five sesquiterpene antibiotics tested and found to inhibit protein synthesis in yeast spheroplasts, trichothecin, trichodermol or trichodermin stabilized polyribosomes whereas, in contrast, verrucarin A or T-2 toxin induced 'run off' of polyribosomes with a corresponding increase in 80S monoribosomes. The effect of fusarenon X on the system could not be determined as the drug failed to enter the cells. 2. [acetyl-14C]Trichodermin bound to yeast polyribosomes with a dissociation constant of 2.10 muM and to yeast 'run off' ribosomes with a dissociation constant of 0.72 muM. 3. Trichothecin, trichodermol, fusarenon X, T-2 toxin and verrucarin A competed with [acetyl-14C]trichodermin for binding to its receptor site on 'run off' ribosomes. The observed competition was quantitatively similar for all drugs tested. In contrast, the five drugs competed to different extents with trichodermin for binding to its receptor site on polyribosomes. Thus trichothecin competed with relative efficiency, whereas verrucarin A competed poorly, and the other drugs occupied intermediate positions between these two extremes. 4. Studies were also carried out with yeast 'run off' ribosomes prepared from both a wild-type strain and a strain resistant to trichodermin. Competition experiments between verrucarin A and [3H]anisomycin indicated that verrucarin A bound to 'run off' ribosomes from the mutant strain less efficiently than to those from the wild-type.
Assuntos
Polirribossomos/metabolismo , Sesquiterpenos/metabolismo , Tricodermina/metabolismo , Anisomicina/metabolismo , Antibacterianos/farmacologia , Ligação Competitiva , Peso Molecular , Receptores de Droga , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/farmacologia , Esferoplastos/metabolismo , Toxina T-2/metabolismo , Tricodermina/análogos & derivados , Tricotecenos/farmacologiaRESUMO
Quantitative binding studies of [G-3H]anisomycin and [acetyl-14C]trichodermin to sensitive and resistant 80-S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y 1661 bind [acetyl-14C]trichodermin with a dissociation constant of 0.99 muM while those from the resistant one (TR1) bind [acetyl-14C]trichodermin with a dissociation constant of 15.4 muM. Similar results are obtained when the binding of [acetyl-14C]trichodermin to Y 166 and TR1 60-S subunits is studied. The mutant TR1 is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G-3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y 166 and TR1 ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin. Trichodermin, trichothecin and fusarenon X inhibit the binding of [G-3H]anisomycin to TR1 ribosomes to a lower extent than to Y 166 ribosomes, suggesting that the resistance of TR1 ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G-3H]anisomycin binding to Y 166 and TR1 ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance. Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60-S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.
Assuntos
Aciltransferases/metabolismo , Antibacterianos/metabolismo , Peptidil Transferases/metabolismo , Receptores de Droga , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Anisomicina/metabolismo , Antifúngicos/metabolismo , Sítios de Ligação , Proteínas Fúngicas/metabolismo , Cinética , Mutação , Fenilalanina/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , MAP Quinase Quinase 4 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Anisomicina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Quinase do Fator 2 de Elongação , Células Epiteliais , Células HeLa , Humanos , MAP Quinase Quinase 1 , MAP Quinase Quinase 6 , Proteína Quinase 12 Ativada por Mitógeno , Proteína Quinase 13 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade por Substrato , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH(2)-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells.