RESUMO
Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKß inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKß and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKß kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKß can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.
Assuntos
Antivirais/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/metabolismo , Quinase I-kappa B/metabolismo , Proteínas não Estruturais Virais/metabolismo , Aedes , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana , Humanos , Mutação , NF-kappa B/metabolismo , Fosforilação , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
Glycine modulates inflammatory processes mediated by macrophages and adipocytes through decreasing the secretion of TNF-α, IL-6, and leptin, while increasing adiponectin. These effects have been associated with the inactivation of NF-κB in response to TNF-α, across an increase of its inhibitor IκB-α in adipocytes. However, glycine upstream mainly influences the IκB kinase (IKK) complex, a multi-protein kinase complex considered a critical point in regulation of the NF-κB pathway; whether that is responsible for the TNF-α-induced phosphorylation of IkB has not been explored. Additionally, although previous studies have described glycine interactions with specific receptors (GlyR) in different immune system cell types, it is currently unknown whether adipocytes present GlyR. In this research, participation of the IKK-α/ß complex in the inhibition of the TNF-α/NF-κB pathway by glycine was evaluated and associated with the synthesis and secretion of inflammatory cytokines in 3T3-L1 adipocytes. Furthermore, we also explored GlyR expression, its localization on the plasmatic membrane, intracellular calcium concentrations [Ca2+]i and strychnine antagonist action over the GlyR in these cells. Glycine decreased the IKK-α/ß complex and the phosphorylation of NF-κB, diminishing the expression and secretion of IL-6 and TNF-α, but increasing that of adiponectin. GlyR expression and its fluorescence in the plasma membrane were increased in the presence of glycine. In addition, glycine decreased [Ca2+]i; whereas strychnineâ¯+â¯glycine treatment inhibited the activation of NF-κB observed with glycine. In conclusion, the reduction of TNF-α and IL-6 and suppression of the TNF-α/NF-κB pathway by glycine may be explained in part by inhibition of the IKK-α/ß complex, with a possible participation of GlyR in 3T3-L1 adipocytes.
Assuntos
Adipócitos/metabolismo , Glicina/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Células 3T3-L1 , Animais , Cálcio/metabolismo , Citocinas/biossíntese , Citocinas/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Fosforilação , Receptores de Glicina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: Recent data show that iNOS has an essential role in ER stress in obesity. However, whether iNOS is sufficient to account for obesity-induced ER stress and Unfolded Protein Response (UPR) has not yet been investigated. In the present study, we used iNOS knockout mice to investigate whether high-fat diet (HFD) can still induce residual ER stress-associated insulin resistance. METHODS: For this purpose, we used the intraperitoneal glucose tolerance test (GTT), euglycemic-hyperinsulinemic clamp, western blotting and qPCR in liver, muscle, and adipose tissue of iNOS KO and control mice on HFD. RESULTS: The results of the present study demonstrated that, in HFD fed mice, iNOS-induced alteration in insulin signaling is an essential mechanism of insulin resistance in muscle, suggesting that iNOS may represent an important target that could be blocked in order to improve insulin sensitivity in this tissue. However, in liver and adipose tissue, the insulin resistance induced by HFD was only partially dependent on iNOS, and, even in the presence of genetic or pharmacological blockade of iNOS, a clear ER stress associated with altered insulin signaling remained evident in these tissues. When this ER stress was blocked pharmacologically, insulin signaling was improved, and a complete recovery of glucose tolerance was achieved. CONCLUSIONS: Taken together, these results reinforce the tissue-specific regulation of insulin signaling in obesity, with iNOS being sufficient to account for insulin resistance in muscle, but in liver and adipose tissue ER stress and insulin resistance can be induced by both iNOS-dependent and iNOS-independent mechanisms.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Dieta Hiperlipídica , Gorduras na Dieta/metabolismo , Insulina/genética , Resistência à Insulina/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/genética , Transdução de Sinais/fisiologia , Resposta a Proteínas não DobradasRESUMO
Inhibitor of nuclear factor κB kinase 2 (IKK2) is suggested to be a potential target for the development of novel anti-inflammatory and anticancer drugs. In this work, we applied structure-based drug design to improve the potency of the inhibitor (E)-N'-(4-nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524, 1 a: IC50 =20 µm). The molecular model built for IKK2 together with the docking methodology employed were able to provide important and consistent information with respect to the structural and chemical inhibitor characteristics that may confer potency to IKK2 inhibitors, providing important guidelines for the development of a new N-acylhydrazone (NAH) derivative. (E)-N'-(4-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzylidene)-2-naphthohydrazide hydrochloride (LASSBio-1829 hydrochloride, 10) is a 7-azaindole NAH able to inhibit IKK2 with an IC50 value of 3.8 µm. LASSBio-1829 hydrochloride was found to be active in several pharmacological inflammation tests in vivo, showing its potential as an anti-inflammatory prototype.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Compostos de Benzilideno/administração & dosagem , Compostos de Benzilideno/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Naftalenos/administração & dosagem , Naftalenos/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Anti-Inflamatórios não Esteroides/química , Compostos de Benzilideno/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Quinase I-kappa B/metabolismo , Modelos Moleculares , Estrutura Molecular , Naftalenos/química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1ß-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzopiranos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Benzopiranos/química , Benzopiranos/isolamento & purificação , Caesalpinia/química , Etnofarmacologia , Genes Reporter/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Estrutura Molecular , NF-kappa B/agonistas , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Receptores de Interleucina-1/agonistas , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , República da Coreia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação/efeitos dos fármacos , Madeira/químicaRESUMO
Cisplatin-based chemotherapy is the standard treatment of choice for head and neck squamous cell carcinoma (HNSCC). The efficiency of platinum-based therapies is directly influenced by the development of tumor resistance. Multiple signaling pathways have been linked to tumor resistance, including activation of nuclear factor kappa B (NFκB). We explore a novel mechanism by which NFκB drives HNSCC resistance through histone modifications. Post-translational modification of histones alters chromatin structure, facilitating the binding of nuclear factors that mediate DNA repair, transcription, and other processes. We found that chemoresistant HNSCC cells with active NFκB signaling respond to chemotherapy by reducing nuclear BRCA1 levels and by promoting histone deacetylation (chromatin compaction). Activation of this molecular signature resulted in impaired DNA damage repair, prolonged accumulation of histone γH2AX and increased genomic instability. We found that pharmacological induction of histone acetylation using HDAC inhibitors prevented NFκB-induced cisplatin resistance. Furthermore, silencing NFκB in HNSCC induced acetylation of tumor histones, resulting in reduced chemoresistance and increased cytotoxicity following cisplatin treatment. Collectively, these findings suggest that epigenetic modifications of HNSCC resulting from NFκB-induced histone modifications constitute a novel molecular mechanism responsible for chemoresistance in HNSCC. Therefore, targeted inhibition of HDAC may be used as a viable therapeutic strategy for disrupting tumor resistance caused by NFκB.
RESUMO
The objective of this study was to investigate the impact of elevated tissue omega-3 (n-3) polyunsaturated fatty acids (PUFA) status on age-related glucose intolerance utilizing the fat-1 transgenic mouse model, which can endogenously synthesize n-3 PUFA from omega-6 (n-6) PUFA. Fat-1 and wild-type mice, maintained on the same dietary regime of a 10% corn oil diet, were tested at two different ages (2 months old and 8 months old) for various glucose homeostasis parameters and related gene expression. The older wild-type mice exhibited significantly increased levels of blood insulin, fasting blood glucose, liver triglycerides, and glucose intolerance, compared to the younger mice, indicating an age-related impairment of glucose homeostasis. In contrast, these age-related changes in glucose metabolism were largely prevented in the older fat-1 mice. Compared to the older wild-type mice, the older fat-1 mice also displayed a lower capacity for gluconeogenesis, as measured by pyruvate tolerance testing (PTT) and hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase). Furthermore, the older fat-1 mice showed a significant decrease in body weight, epididymal fat mass, inflammatory activity (NFκ-B and p-IκB expression), and hepatic lipogenesis (acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression), as well as increased peroxisomal activity (70-kDa peroxisomal membrane protein (PMP70) and acyl-CoA oxidase1 (ACOX1) expression). Altogether, the older fat-1 mice exhibit improved glucose homeostasis in comparison to the older wild-type mice. These findings support the beneficial effects of elevated tissue n-3 fatty acid status in the prevention and treatment of age-related chronic metabolic diseases.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Intolerância à Glucose/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Fatores Etários , Animais , Glicemia/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ácidos Graxos Dessaturases/genética , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Expressão Gênica , Gluconeogênese/genética , Glucose/metabolismo , Intolerância à Glucose/genética , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Homeostase/genética , Immunoblotting , Insulina/sangue , Lipogênese/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.