RESUMO
The evolutionarily conserved extracellular signal-regulated kinase 2 (ERK2) is involved in regulating cellular signaling in both normal and pathological conditions. ERK2 expression is critical for human development, while hyperactivation is a major factor in tumor progression. Up to now, there have been no approved inhibitors that target ERK2, and as such, here we report on screening of a naturally occurring plant-based anticancerous compound-activity-target (NPACT) database for prospective ERK2 inhibitors. More than 1,500 phytochemicals were screened using in-silico molecular docking and molecular dynamics (MD) approaches. NPACT compounds with a docking score lower than a co-crystallized LHZ inhibitor (calc.-10.5 kcal/mol) were subjected to MD simulations. Binding energies (ΔGbinding) of inhibitor-ERK2 complexes over the MD course were estimated using an MM-GBSA approach. Based on MM-GBSA//100 ns MD simulations, the steroid zhankuic acid C (NPACT01034) demonstrated greater binding affinity against ERK2 protein than LHZ, with ΔGbinding values of -50.0 and -47.7 kcal/mol, respectively. Structural and energetical analyses throughout the MD course demonstrated stabilization of zhankuic acid C complexed with ERK2 protein. The anticipated ADMET properties of zhankuic acid C indicated minimal toxicity. Moreover, in-silico evaluation of fourteen ERK2 inhibitors in clinical trials demonstrated the higher binding affinity of zhankuic acid C towards ERK2 protein.
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Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.
Assuntos
Cromossomos Humanos/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mitose , Cromossomos Humanos/genética , Fosfatases de Especificidade Dupla/genética , Células HCT116 , Células HeLa , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Mutação , Fosforilação/genéticaRESUMO
Posttraumatic stress disorder subjects usually show impaired recall of extinction memory, leading to extinguished fear relapses. However, little is known about the neural mechanisms underlying the impaired recall of extinction memory. We show here that the activity of dorsal hippocampus (dHPC) to infralimbic (IL) cortex circuit is essential for the recall of fear extinction memory in male mice. There were functional neural projections from the dHPC to IL. Using optogenetic manipulations, we observed that silencing the activity of dHPC-IL circuit inhibited recall of extinction memory while stimulating the activity of dHPC-IL circuit facilitated recall of extinction memory. "Impairment of extinction consolidation caused by" conditional deletion of extracellular signal-regulated kinase 2 (ERK2) in the IL prevented the dHPC-IL circuit-mediated recall of extinction memory. Moreover, silencing the dHPC-IL circuit abolished the effect of intra-IL microinjection of ERK enhancer on the recall of extinction memory. Together, we identify a dHPC to IL circuit that mediates the recall of extinction memory, and our data suggest that the dysfunction of dHPC-IL circuit and/or impaired extinction consolidation may contribute to extinguished fear relapses.
Assuntos
Extinção Psicológica/fisiologia , Hipocampo/fisiologia , Memória/fisiologia , Vias Neurais/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Condicionamento Clássico , Masculino , Camundongos Endogâmicos C57BL , Transtornos de Estresse Pós-Traumáticos/fisiopatologiaRESUMO
Bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) is a significant lung morbidity of infants, and disrupted lung angiogenesis is a hallmark of this disease. We observed that extracellular signal-regulated kinases (ERK) 1/2 support angiogenesis in vitro, and hyperoxia activates ERK1/2 in fetal human pulmonary microvascular endothelial cells (HPMECs) and in neonatal murine lungs; however, their role in experimental BPD and PH is unknown. Therefore, we hypothesized that Tie2 Cre-mediated deficiency of ERK2 in the endothelial cells of neonatal murine lungs would potentiate hyperoxia-induced BPD and PH. We initially determined the role of ERK2 in in vitro angiogenesis using fetal HPMECs. To disrupt endothelial ERK2 signaling in the lungs, we decreased ERK2 expression by breeding ERK2flox/flox mice with Tie-Cre mice. One-day-old endothelial ERK2-sufficient (eERK2+/+) or -deficient (eERK2+/-) mice were exposed to normoxia or hyperoxia (FiO2 70%) for 14 d. We then performed lung morphometry, gene and protein expression studies, and echocardiography to determine the extent of inflammation, oxidative stress, and development of lungs and PH. The knockdown of ERK2 in HPMECs decreased in vitro angiogenesis. Hyperoxia increased lung inflammation and oxidative stress, decreased lung angiogenesis and alveolarization, and induced PH in neonatal mice; however, these effects were augmented in the presence of Tie2-Cre mediated endothelial ERK2 deficiency. Therefore, we conclude that endothelial ERK2 signaling is necessary to mitigate hyperoxia-induced experimental BPD and PH in neonatal mice. Our results indicate that endothelial ERK2 is a potential therapeutic target for the management of BPD and PH in infants.
Assuntos
Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Hipertensão Pulmonar/metabolismo , Integrases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/deficiência , Proteína Quinase 1 Ativada por Mitógeno/genética , Receptor TIE-2/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/patologia , Células Endoteliais/metabolismo , Humanos , Hiperóxia/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/metabolismo , Pulmão/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Pneumonia/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
A number of natural phytochemicals have anti-photoaging properties that appear to be mediated through the inhibition of matrix metalloproteinase-1 (MMP-1) expression, but their direct target molecule(s) and mechanism(s) remain unclear. We investigated the effect of naringenin, a major flavonoid found in citrus, on UVB-induced MMP-1 expression and identified its direct target. The HaCaT human skin keratinocyte cell line and 3-dimensional (3-D) human skin equivalent cultures were treated or not treated with naringenin for 1 hr before exposure to UVB. The mechanism and target(s) of naringenin were analysed by kinase assay and multiplex molecular assays. Dorsal skins of hairless mice were exposed to UVB 3 times per week, with a dose of irradiation that was increased weekly by 1 minimal erythema dose (MED; 45 mJ/cm(2)) to 4 MED over 15 weeks. Wrinkle formation, water loss and water content were then assessed. Naringenin suppressed UVB-induced MMP-1 expression and AP-1 activity, and strongly suppressed UVB-induced phosphorylation of Fos-related antigen (FRA)-1 at Ser265. Importantly, UVB irradiation-induced FRA1 protein stability was reduced by treatment with naringenin, as well as with a mitogen-activated protein kinase (MEK) inhibitor. Naringenin significantly suppressed UVB-induced extracellular signal-regulated kinase 2 (ERK2) activity and subsequently attenuated UVB-induced phosphorylation of p90(RSK) by competitively binding with ATP. Constitutively active MEK (CA-MEK) increased FRA1 phosphorylation and expression and also induced MMP-1 expression, whereas dominant-negative ERK2 (DN-ERK2) had opposite effects. U0126, a MEK inhibitor, also decreased FRA1 phosphorylation and expression as well as MMP-1 expression. The photoaging data obtained from mice clearly demonstrated that naringenin significantly inhibited UVB-induced wrinkle formation, trans-epidermal water loss and MMP-13 expression. Naringenin exerts potent anti-photoaging effects by suppressing ERK2 activity and decreasing FRA1 stability, followed by down-regulation of AP-1 transactivation and MMP-1 expression.
Assuntos
Flavanonas/farmacologia , Queratinócitos/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Envelhecimento da Pele/efeitos dos fármacos , Protetores Solares/farmacologia , Raios Ultravioleta/efeitos adversos , Animais , Butadienos/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Genes Reporter , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Luciferases/genética , Luciferases/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Pelados , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Envelhecimento da Pele/genética , Envelhecimento da Pele/patologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Água/metabolismoRESUMO
20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inflamação/prevenção & controle , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Bovinos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citocromo P-450 CYP4A/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genótipo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Inflamação/enzimologia , Inflamação/genética , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Mutantes , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias Musculares/enzimologia , Músculo Liso Vascular/enzimologia , Mutação , Miócitos de Músculo Liso/enzimologia , Fenótipo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Farming of Eriocheir sinensis was seriously threaten by the infection of opportunistic pathogens, especially the gram-negative bacterium. In this paper, we analyzed the sequence of extracellular signal-regulated kinases 2 (ERK2) of E. sinensis (EsERK2) and its expression levels after challenge with LPS and Aeromonas hydrophila in both in vivo and in vitro examination. The full-length cDNA sequence of EsERK2 was 2455 bp in size with an open reading frame (ORF) of 1095 bp, encoding the protein of 365 amino acids. It owned a predicted molecular mass of 42.4 kDa and a theoretical isoelectric point (pI) of 5.93. EsERK2 was distributed in all examined tissues including haemocyte, gonad, hepatopancreas, gill, muscle heart, stomach and intestine, but its expression level was significantly higher in hepatopancreas than it in other examined tissues. The expression level of EsERK2 increased significantly after LPS challenge at 2 h (P < 0.05), and then gradually increased and reached highest at 16 h. However, its expression level decreased significantly after A. hydrophila challenge at 4 h, and then gradually decreased till 24 h (P < 0.05), and returned to its initial value at 36 h. According to the immunofluorescence assay and western blotting assay, EsERK2 was found to be distributed mainly in cytoplasm of haemocyte, and its expression level showed a prominent boost in primary cultured haemocytes after challenge with LPS and A. hydrophila in vitro. These results indicated that the expression of EsERK2 was sensitive to the exterior stimulants and its encoding protein might be associated with the signaling transduction in response to exterior pathogens in E. sinensis.
Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica , Proteína Quinase 1 Ativada por Mitógeno/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Braquiúros/enzimologia , Braquiúros/microbiologia , DNA Complementar/genética , DNA Complementar/metabolismo , Lipopolissacarídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Alinhamento de SequênciaRESUMO
Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.
Assuntos
Baculoviridae/genética , Clonagem Molecular/métodos , Proteína Quinase 1 Ativada por Mitógeno/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Animais , Baculoviridae/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Engenharia Genética , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/isolamento & purificação , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Plasmídeos/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Células Sf9 , SpodopteraRESUMO
Extracellular signal-regulated kinase 2 (ERK-2) is a serine/threonine protein kinase in eukaryotic cells and belongs to the mitogen-activated protein kinase (MAPK) family. An activated form of ERK-2 phosphorylates substrates in the nucleus or cytoplasm and causes specific proteins to be expressed or activated, regulating cell proliferation, differentiation and other functions. Caffeic acid (3,4 - dihydroxy cinnamic acid), as previously reported, directly interacts with ERK-2 and reduces its effects in vitro. It is also reported to have a variety of pharmacological effects, including anti-inflammatory, immunomodulatory, antioxidant and anticancer activities. In the current study, a deep-learning protocol was employed to develop effective 100 compounds by modifying the chemical structure of DHC to improve its inhibitory performance against ERK-2. Calculations of physicochemical properties for those compounds revealed that 20 compounds had drug scores better than DHC (≥ 80%). Following that, molecular docking calculations were performed on the selected compounds and DHC. The obtained data revealed that five compounds had docking scores better than DHC (≥ -5.9 kcal/mol). Moreover, data from molecular mechanics and the Poisson - Boltzmann surface area (MM/PBSA) binding energy over 200 ns MD simulation confirmed that Cmd-1 and Cmd-4 exhibited higher stability with ΔGbinding of -40.8 and -49.1 kcal/mol, respectively, which is better than DHC (-35.1 kcal/mol). Finally, various energetic and structural studies showed the high stability of the two generated compounds within the active site of ERK-2. This study highlights the potential use of Cmd-1 and Cmd-4 as promising anti-ERK-2 drug candidates.Communicated by Ramaswamy H. Sarma.
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Acute kidney injury (AKI) is a refractory clinical syndrome with limited effective treatments. Amid AKI, activation of the extracellular signal-regulated kinase (ERK) cascade plays a critical role in promoting kidney repair and regeneration. However, a mature ERK agonist in treating kidney disease remains lacking. This study identified limonin, a member of the class of compounds known as furanolactones, as a natural ERK2 activator. Employing a multidisciplinary approach, we systemically dissected how limonin mitigates AKI. Compared to vehicles, pretreatment of limonin significantly preserved kidney functions after ischemic AKI. We revealed that ERK2 is a significant protein linked to the limonin's active binding sites through structural analysis. The molecular docking study showed a high binding affinity between limonin and ERK2, which was confirmed by the cellular thermal shift assay and microscale thermophoresis. Mechanistically, we further validated that limonin promoted tubular cell proliferation and reduced cell apoptosis after AKI by activating ERK signaling pathway in vivo. In vitro and ex vivo, blockade of ERK abolished limonin's capacity of preventing tubular cell death under hypoxia stress. Our results indicated that limonin is a novel ERK2 activator with strong translational potential in preventing or mitigating AKI.
Assuntos
Injúria Renal Aguda , Limoninas , Humanos , Limoninas/farmacologia , Limoninas/uso terapêutico , Simulação de Acoplamento Molecular , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Rim/metabolismo , Apoptose , MAP Quinases Reguladas por Sinal ExtracelularRESUMO
Extracellular signal-regulated kinase (ERK) is the culmination of a mitogen-activated protein kinase cascade that regulates cellular processes like proliferation, migration, and survival. Consequently, abnormal ERK signaling often plays a role in the tumorigenesis and metastasis of numerous cancers. ERK inhibition is a sought-after treatment for cancers, especially since clinically approved drugs that target signaling upstream of ERK often induce acquired resistance. Furthermore, the ERK2 isoform may have a differential role in various cancers from the other canonical isoform, ERK1. We demonstrate that small molecules can inhibit ERK2 catalytic and noncatalytic functions by binding to the D-recruitment site (DRS), a protein-protein interaction site distal to the enzyme active site. Using a fluorescence anisotropy-based high-throughput screening, we identify compounds that bind to the DRS and exhibit dose-dependent inhibition of ERK2 activity and ERK2 phosphorylation. We characterize the dose-dependent potency of ERK2 inhibitors using fluorescence anisotropy-based binding assays, fluorescence-based ERK2 substrate phosphorylation assays, and in vitro ERK2 activation assays. In our example, the binding of a DRS inhibitor can be prevented by mutating the DRS residue Cys-159 to serine, indicating that this residue is essential for the interaction. Resulting inhibitors from this process can be assessed in cellular and in vivo experiments for inhibition of ERK signaling and can be evaluated as potential cancer drugs.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Transdução de Sinais , Fosforilação , Transdução de Sinais/fisiologia , Processamento de Proteína Pós-Traducional , Isoformas de ProteínasRESUMO
Background Metabolic syndrome is characterized by insulin resistance, which impairs intracellular signaling pathways and endothelial NO bioactivity, leading to cardiovascular complications. Extracellular signal-regulated kinase (ERK) is a major component of insulin signaling cascades that can be activated by many vasoactive peptides, hormones, and cytokines that are elevated in metabolic syndrome. The aim of this study was to clarify the role of endothelial ERK2 in vivo on NO bioactivity and insulin resistance in a mouse model of metabolic syndrome. Methods and Results Control and endothelial-specific ERK2 knockout mice were fed a high-fat/high-sucrose diet (HFHSD) for 24 weeks. Systolic blood pressure, endothelial function, and glucose metabolism were investigated. Systolic blood pressure was lowered with increased NO products and decreased thromboxane A2/prostanoid (TP) products in HFHSD-fed ERK2 knockout mice, and Nω-nitro-l-arginine methyl ester (L-NAME) increased it to the levels observed in HFHSD-fed controls. Acetylcholine-induced relaxation of aortic rings was increased, and aortic superoxide level was lowered in HFHSD-fed ERK2 knockout mice. S18886, an antagonist of the TP receptor, improved endothelial function and decreased superoxide level only in the rings from HFHSD-fed controls. Glucose intolerance and the impaired insulin sensitivity were blunted in HFHSD-fed ERK2 knockout mice without changes in body weight. In vivo, S18886 improved endothelial dysfunction, systolic blood pressure, fasting serum glucose and insulin levels, and suppressed nonalcoholic fatty liver disease scores only in HFHSD-fed controls. Conclusions Endothelial ERK2 increased superoxide level and decreased NO bioactivity, resulting in the deterioration of endothelial function, insulin resistance, and steatohepatitis, which were improved by a TP receptor antagonist, in a mouse model of metabolic syndrome.
Assuntos
Resistência à Insulina , Síndrome Metabólica , Animais , Camundongos , Síndrome Metabólica/genética , MAP Quinases Reguladas por Sinal Extracelular , Receptores de Tromboxano A2 e Prostaglandina H2 , Tromboxano A2 , Prostaglandinas , Camundongos Knockout , InsulinaRESUMO
Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. ß-arrestin1 and ß-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno , beta-Arrestina 1 , beta-Arrestina 2 , Proteína Quinase 1 Ativada por Mitógeno/química , Ligação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X , beta-Arrestina 1/química , beta-Arrestina 2/químicaRESUMO
The cell proliferation effect of S-allyl-L-cysteine (SAC) and its mechanisms were examined in primary cultures of adult rat hepatocytes. In serum-free cultivation, SAC (10-6 M)-stimulated hepatocytes showed significant proliferation compared to control at 5-h culture; the effect was dependent on the culture time and the dose of SAC (EC50 value 8.58 × 10-8 M). In addition, SAC-stimulated hepatocytes significantly increased mRNA expression levels of c-Myc and c-Fos at 1 h and cyclin B1 at 3.5 and 4 h, respectively. In contrast, alliin and allicin, structural analogs of SAC, did not show these effects observed with SAC. The SAC-induced hepatocyte proliferation effects were completely suppressed by monoclonal antibodies against growth hormone receptor and insulin-like growth factor type-I (IGF-I) receptor, respectively. Furthermore, the Janus kinase 2 (JAK2) inhibitor TG101209, phospholipase C (PLC) inhibitor U-73122, IGF-I receptor tyrosine kinase (RTK) inhibitor AG538, PI3 kinase inhibitor LY294002, MEK inhibitor PD98059, and mTOR inhibitor rapamycin completely suppressed the SAC-induced hepatocyte proliferation. JAK2 (p125 kDa) phosphorylation in cultured hepatocytes peaked 5 min after SAC stimulation. SAC-induced IGF-I RTK (p95 kDa) and ERK2 (p42 kDa) phosphorylation had slower rises than JAK2, peaking at 20 and 30 min, respectively. These results indicate that SAC promoted cell proliferation by growth hormone receptor/JAK2/PLC pathway activation followed by activation of the IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.
Assuntos
Cisteína/análogos & derivados , Hepatócitos , Fator de Crescimento Insulin-Like I , Janus Quinase 2 , Proteína Quinase 1 Ativada por Mitógeno , Receptor IGF Tipo 1 , Animais , Proliferação de Células/efeitos dos fármacos , Cisteína/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Janus Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Ratos , Receptor IGF Tipo 1/metabolismo , Receptores da Somatotropina/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Interleukin (IL)-8 and extracellular signal-regulated kinase (ERK) 2 play key roles in tumor progression, but the relationship between IL-8 and/or ERK2 expression in hepatocellular carcinoma (HCC) tissues and postoperative recurrence or survival is unclear. The expression levels of IL-8 and ERK2 in both HCC tissues and non-tumor liver tissues were analyzed using the Oncomine™ database and immunohistochemistry assay. Reverse transcription-quantitative PCR was then used to evaluate the expression levels of IL-8 and ERK2 in the tumor tissues of 67 patients with HCC undergoing radical hepatectomy. Pearson's correlation, Kaplan-Meier, Cox univariate and multivariate survival analyses were utilized to determine the correlation between IL-8 and ERK2 expression in HCC tissues, and their potential prognostic significance. As indicated by the data from the Oncomine™ database, and the patient samples, IL-8 and ERK2 were expressed at significantly higher levels in HCC tissues than in non-tumor liver tissues (P<0.05). The rates of high IL-8 and ERK2 expression in HCC tissues were 43.28 (29/67) and 34.33% (23/67), respectively, and the IL-8 and ERK2 expression levels were positively correlated (r=0.764; P<0.001). Both ERK2 expression and IL-8/ERK2 co-expression were significantly associated with tumor size and differentiation (P<0.05). Additionally, high expression levels of IL-8, ERK2 and IL-8/ERK2 co-expression were all significantly associated with poor overall survival (OS; P<0.05) and disease-free survival (DFS; P<0.05). Multivariate Cox regression analysis also showed that high expression levels of IL-8, ERK2, and IL-8 and ERK2 were independent prognostic factors for OS and DFS (P<0.05). The results of the present study indicate a significant increase in the risk of recurrence and mortality in HCC patients with high expression levels of IL-8 and/or ERK2, compared with patients with low expression. Therefore, IL-8 and ERK2 may be predictors of postoperative prognosis in patients with HCC.
RESUMO
OBJECTIVE: To investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model. METHODS: The intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined. RESULTS: Compared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01). CONCLUSION: Rut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways.
Assuntos
Artérias Carótidas/patologia , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/patologia , Alcaloides Indólicos/farmacologia , Alcaloides Indólicos/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Túnica Íntima/patologia , Actinas/metabolismo , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/genética , GMP Cíclico/sangue , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperplasia , Masculino , Óxido Nítrico/sangue , Fosforilação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Túnica Íntima/efeitos dos fármacosRESUMO
Molecular dynamics (MD) simulations coupled with principal component (PC) analysis were carried out to study functional roles of Mg2+ binding to extracellular signal-regulated kinase 2 (ERK2). The results suggest that Mg2+ binding heavily decreases eigenvalue of the first principal component and totally inhibits motion strength of ERK2, which favors stabilization of ERK2 structure. Binding free energy predictions indicate that Mg2+ binding produces an important effect on binding ability of adenosine triphosphate (ATP) to ERK2 and strengthens the ATP binding. The calculations of residue-based free energy decomposition show that lack of Mg2+ weakens interactions between the hydrophobic rings of ATP and five residues I29, V37, A50, L105, and L154. Hydrogen bond analyses also prove that Mg2+ binding increases occupancies of hydrogen bonds formed between ATP and residues K52, Q103, D104, and M106. We expect that this study can provide a significant theoretical hint for designs of anticancer drugs targeting ERK2.
Assuntos
Trifosfato de Adenosina/química , Magnésio/química , Proteína Quinase 1 Ativada por Mitógeno/química , Simulação de Dinâmica Molecular , Sítios de Ligação , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/uso terapêutico , Neoplasias/tratamento farmacológico , Análise de Componente Principal , Ligação ProteicaRESUMO
Extracellular signal-regulated kinase 2 is a promising target for designs and development of anticancer drugs. Molecular dynamics simulations and molecular mechanics Poisson-Boltzmann method were applied to study binding difference of ADP and ATP to extracellular signal-regulated kinase 2. The results prove that the binding ability of ATP to extracellular signal-regulated kinase 2 is stronger than that of ADP. Principal component analysis performed by using molecular dynamics trajectories suggests that binding of ADP and ATP to extracellular signal-regulated kinase 2 change motion directions of two helices α1 and α2. Residue-based free energy decomposition method was adopted to calculate contributions of separate residues to associations of ADP and ATP with extracellular signal-regulated kinase 2. The results show that ADP and ATP produce strong CH-π interactions with five residues Ile29, Val37, Ala50, Leu105, and Leu154. In addition, five hydrogen bonding interactions of ADP and ATP with residues Lys52, Gln103, Asp104, and Met106 also stabilize bindings of ADP and ATP to extracellular signal-regulated kinase 2. Overall, the CH-π interactions of ATP with five residues Ile29, Val37, Ala50, Leu105, and Leu154 are stronger than ADP. This study is expected to contribute a significant theoretical hint for designs of anticancer drugs targeting extracellular signal-regulated kinase 2.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sítios de Ligação , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Simulação de Dinâmica Molecular , Análise de Componente PrincipalRESUMO
Dietary intakes of taurine and zinc are associated with decreased risk of liver disease. In this study, solid dispersions (SDs) of a taurine zinc complex on hepatic injury were examined in vitro using the immortalized human hepatocyte cell line L02 and in a rat model of bile duct ligation. Sham-operated and bile duct ligated Sprague-Dawley rats were treated with the vehicle alone or taurine zinc (40, 80, 160mg/kg) for 17days. Bile duct ligation significantly increased blood lipid levels, and promoted hepatocyte apoptosis, inflammation and compensatory biliary proliferation. In vitro, incubation with bile significantly reduced L02 cell viability; this effect was significantly attenuated by pretreatment with SP600125 (a JNK inhibitor) and enhanced when co-incubated with taurine zinc SDs. In vivo, administration of taurine zinc SDs decreased serum alanine aminotransferase and aspartate aminotransferase activities in a dose-dependent manner and attenuated the increases in serum total bilirubin, total cholesterol and low density lipoprotein cholesterol levels after bile duct ligation. Additionally, taurine zinc SDs downregulated the expression of interleukin-1ß and inhibited the phosphorylation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase2 (ERK2) in the liver after bile duct ligation. Moreover, taurine zinc SDs had more potent blood lipid regulatory and anti-apoptotic effects than the physical mixture of taurine and zinc acetate. Therefore, we speculate that taurine zinc SDs protect liver function at least in part via a mechanism linked to reduce phosphorylation of JNK and ERK2, which suppresses inflammation, apoptosis and cholangiocyte proliferation during cholestasis.