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Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. Here, we report a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, its phosphorylation at Thr287 by ßCaMKII protects the Ca(2+)/CaM signal, and CaN triggers its nuclear translocation. Both ßCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, ßCaMKII, and CaN in multiple neuropsychiatric disorders.
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Calmodulina/metabolismo , Núcleo Celular/metabolismo , Neurônios/metabolismo , Fosforilação , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
New agents are urgently needed for the therapeutic treatment of Staphylococcus aureus infections. In that regard, S. aureus RNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify small-molecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series of in vitro and cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found that S. aureus RnpA does indeed participate in 5'-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities both in vitro and within S. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect the in vitro activities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities.
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Antibacterianos/farmacologia , RNA Bacteriano/efeitos dos fármacos , RNA de Transferência/efeitos dos fármacos , Ribonuclease P/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Hidrazinas/farmacologia , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas , Tioureia/análogos & derivados , Tioureia/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Mercury vapor poses a known health risk with no clearly established safe level of exposure. Consequently there is debate over whether the level of prolonged exposure to mercury vapor from dental amalgam fillings, combining approximately 50% mercury with other metals, is sufficiently high to represent a risk to health. The objective of our study is to determine if mercury exposure from amalgam fillings is associated with risk of adverse health effects. METHODS: In a large longitudinal non-blind sample of participants from a preventative health program in Calgary, Canada we compared number of amalgam fillings, urine mercury measures and changes in 14 self-reported health symptoms, proposed to be mercury dependent sub-clinical measures of mental and physical health. The likelihood of change over one year in a sample of persons who had their fillings removed was compared to a sample of persons who had not had their fillings removed. We use non-parametric statistical tests to determine if differences in urine mercury were statistically significant between sample groups. Logistic regression models were used to estimate the likelihood of observing symptom improvement or worsening in the sample groups. RESULTS: At baseline, individuals with dental amalgam fillings have double the measured urine mercury compared to a control group of persons who have never had amalgam fillings. Removal of amalgam fillings decreases measured urine mercury to levels in persons without amalgam fillings. Although urine mercury levels in our sample are considered by Health Canada to be too low to pose health risks, removal of amalgam fillings reduced the likelihood of self-reported symptom deterioration and increased the likelihood of symptom improvement in comparison to people who retained their amalgam fillings. CONCLUSIONS: Our findings suggest that mercury exposure from amalgam fillings adversely impact health and therefore are a health risk. The use of safer alternative materials for dental fillings should be encouraged to avoid the increased risk of health deterioration associated with unnecessary exposure to mercury.
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Amálgama Dentário/efeitos adversos , Mercúrio/toxicidade , Adulto , Canadá/epidemiologia , Monitoramento Ambiental , Feminino , Nível de Saúde , Humanos , Masculino , Mercúrio/urina , Pessoa de Meia-Idade , Medição de Risco , AutorrelatoRESUMO
BACKGROUND: DVT is the main cause of death in hospitalized patients and thromboprophylaxis is the only way to prevent these deaths. International recommendations suggested that active monitoring of DVT/PE prophylaxis can improve the efficacy in Hospitals. METHODS: We performed a cohort study in three consecutives periods to evaluate DVT prophylaxis in 388 adults hospitalized in a General Hospital. RESULTS: 85% of the population had high risk factors for DVT. Thromboprophylaxis was in accordance with local and International guidelines (ACCP 2008) in 72.7% and 86% of the patients respectively. No significant difference could be founded between clinical and surgical patients. One every 10 patients received higher prophylaxis than suggested by guidelines and two out of ten received deficient or no prophylaxis. The worst 2 groups of patients were those with moderate/low risk of DVT and the group with a contraindication to pharmacologic prophylaxis. We observed a progressive improvement of the DVT prophylaxis in the 3 periods of evaluation. CONCLUSIONS: Although the rate of recommended thromboprophylaxis is higher than many other reports in the region we still have some areas where we need to improve. Regular audits like these are very helpful to find out what specific areas of the hospital needs some careful attention in order to have a better quality of assistance.
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BACKGROUND: William Osler was generally regarded as the greatest and most respected physician of his time. This paper describes Osler's life, his philosophy and views. He was an outstanding clinician who emphasized bedside teaching and observation. He possessed an extraordinary charm that inspired many others. As Professor of Medicine at four institutions in three countries, he was a great influence on medical education. He was a prolific writer, and his textbook became the most popular and widely read treatise on medicine in the world. He also was a medical historian, a classical scholar, and an avid bibliophile. He emphasized the value of hard work and ongoing education. His compassion and concern for patients and colleagues reflected his personality. We summarize Osler's descriptions, and some of his aphorisms. His wisdom is as relevant now, as it was in his time. Osler blended the art and science of Medicine perhaps better than anyone else, and remains a valuable role model for students and physicians more than ninety two years after his death.
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Educação Médica/história , Aforismos e Provérbios como Assunto , Baltimore , Canadá , Inglaterra , História do Século XIX , História do Século XX , Philadelphia , FilateliaRESUMO
The impairment of hippocampal neurogenesis has been linked to the pathogenesis of neurological disorders from chronic neurodegenerative disease to the progressive cognitive impairment of children who receive brain irradiation. Numerous studies provide evidence that inflammation downregulates neurogenesis, with multiple factors contributing to this impairment. Although mitochondria are one of the primary targets of inflammatory injury, the role of mitochondrial function in the modulation of neurogenesis remains relatively unstudied. In this study, we used neurosphere-derived cells to show that immature doublecortin (Dcx)-positive neurons are uniquely sensitive to mitochondrial inhibition, demonstrating rapid loss of mitochondrial potential and cell viability compared with glial cells and more mature neurons. Mitochondrial inhibition for 24 h produced no significant changes in astrocyte or oligodendrocyte viability, but reduced viability of mature neurons by 30%, and reduced survival of Dcx(+) cells by 60%. We demonstrate that protection of mitochondrial function with mitochondrial metabolites or the mitochondrial chaperone mtHsp75/mortalin partially reverses the inflammation-associated impairment of neurogenesis in vitro and in irradiated mice in vivo. Our findings highlight mitochondrial mechanisms involved in neurogenesis and indicate mitochondria as a potential target for protective strategies to prevent the impairment of neurogenesis by inflammation.
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Metabolismo Energético/fisiologia , Mediadores da Inflamação/fisiologia , Mitocôndrias/patologia , Inibição Neural/fisiologia , Neurogênese/fisiologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Metabolismo Energético/genética , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/fisiologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/biossíntese , Mitocôndrias/genética , Mitocôndrias/fisiologia , Inibição Neural/genética , Neurogênese/genética , Neurônios/metabolismo , Neurônios/patologia , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/biossíntese , Tiamina/administração & dosagemRESUMO
BACKGROUND: Activation of the NF-κB transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. Its activation is dynamic and is part of a network of biochemical species with multiple feedback regulatory mechanisms. Mathematical modeling, which has been instrumental for understanding the NF-κB response in other cell types, offers a valuable tool to investigate the regulation of NF-κB activation in microglia at a systems level. RESULTS: We quantify the dynamic response of NF-κB activation and activation of the upstream kinase IKK using ELISA measurements of a microglial cell line following treatment with the pro-inflammatory cytokine TNFα. A new mathematical model is developed based on these data sets using a modular procedure that exploits the feedback structure of the network. We show that the new model requires previously unmodeled dynamics involved in the stimulus-induced degradation of the inhibitor IκBα in order to properly describe microglial NF-κB activation in a statistically consistent manner. This suggests a more prominent role for the ubiquitin-proteasome system in regulating the activation of NF-κB to inflammatory stimuli. We also find that the introduction of nonlinearities in the kinetics of IKK activation and inactivation is essential for proper characterization of transient IKK activity and corresponds to known biological mechanisms. Numerical analyses of the model highlight key regulators of the microglial NF-κB response, as well as those governing IKK activation. Results illustrate the dynamic regulatory mechanisms and the robust yet fragile nature of the negative feedback regulated network. CONCLUSIONS: We have developed a new mathematical model that incorporates previously unmodeled dynamics to characterize the dynamic response of the NF-κB signaling network in microglia. This model is the first of its kind for microglia and provides a tool for the quantitative, systems level study the dynamic cellular response to inflammatory stimuli.
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Isquemia Encefálica/metabolismo , Microglia/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Isquemia Encefálica/patologia , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Camundongos , NF-kappa B/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We report herein the discovery of quinazolindiones as potent and selective tankyrase inhibitors. Elucidation of the structure-activity relationship of the lead compound 1g led to truncated analogues that have good potency in cells, pharmacokinetic (PK) properties, and excellent selectivity. Compound 21 exhibited excellent potencies in cells and proliferation studies, good selectivity, in vitro activities, and an excellent PK profile. Compound 21 also inhibited H292 xenograft tumor growth in nude mice. The synthesis, biological, pharmacokinetic, in vivo efficacy studies, and safety profiles of compounds are presented.
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Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well-studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter-1 immunoreactivity at 5-h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation.
Assuntos
Astrócitos/fisiologia , Isquemia Encefálica/fisiopatologia , Região CA1 Hipocampal/fisiopatologia , Proteínas de Choque Térmico HSP72/metabolismo , Neurônios/fisiologia , Superóxido Dismutase/metabolismo , Animais , Região CA1 Hipocampal/patologia , Hipóxia Celular/fisiologia , Células Cultivadas , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteínas de Choque Térmico HSP72/genética , Humanos , Masculino , Camundongos , Neurônios/patologia , Estresse Oxidativo/fisiologia , Regiões Promotoras Genéticas , Prosencéfalo/patologia , Prosencéfalo/fisiopatologia , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/genética , TransfecçãoRESUMO
Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) is a key mediator of innate immunity. IRAK4 overactivation is linked with several autoimmune diseases. To date, many IRAK4 inhibitors have been developed to block the protein's kinase activity with the most advanced reaching Phase II clinical trials. Nevertheless, several reports suggest kinase activity is not disease-relevant in certain cell types, so removing scaffolding signaling in addition to IRAK4 kinase activity may offer a better therapeutic outcome. Herein, we describe the design and synthesis of an IRAK4 Proteolysis Targeted Chimera (PROTAC). We show that IRAK4 degradation induced by compound 9 leads to the inhibition of multiple cytokines in PBMCs. However, in IL-1ß stimulated human dermal fibroblasts, inhibition of IL-6 and TNF-α release was not observed despite IRAK4 degradation. Nonetheless, the possibility of targeting both IRAK4 kinase and scaffolding function could potentially lead to new therapeutic opportunities to treat autoimmune, inflammatory, and oncological diseases.
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Mitochondrial heat shock protein 70 (mtHsp70/Hsp75/Grp75/mortalin/TRAP-1/PBP74) is an essential mitochondrial chaperone and a member of the heat shock protein 70 (HSP70) family. Although many studies have shown the protective properties of overexpression of the cytosolic inducible member of the HSP70 family, Hsp72, few studies have investigated the protective potential of Hsp75 against ischemic injury. Mitochondria are one of the primary targets of ischemic injury in astrocytes. In this study, we analyzed the effects of Hsp75 overexpression on cellular levels of reactive oxygen species (ROS), mitochondrial membrane potential, ATP levels, and viability during the ischemia-like conditions of oxygen-glucose deprivation (OGD) or glucose deprivation (GD) in primary astrocytic cultures. We show that Hsp75 overexpression decreases ROS production and preserves mitochondrial membrane potential during GD, and preserves ATP levels and cell viability during OGD. These findings indicate that Hsp75 can provide protection against ischemia-like in vitro injury and suggest that it should be further studied as a potential candidate for protection against ischemic injury.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Peróxido de Carbamida , Sobrevivência Celular/fisiologia , Células Cultivadas , Combinação de Medicamentos , Expressão Gênica/fisiologia , Glucose/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Humanos , Técnicas In Vitro , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Peróxidos/farmacologia , Transdução Genética , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Although heat shock proteins have been studied for decades, new intracellular and extracellular functions in a variety of diseases continue to be discovered. Heat shock proteins function within networks of interacting proteins; they can alter cellular physiology rapidly in response to stress without requiring new protein synthesis. This review focuses on the heat shock protein 70 family and considers especially the functions of the inducible member, heat shock protein 72, in the setting of cerebral ischemia. In general, inhibiting apoptotic signaling at multiple points and up-regulating survival signaling, heat shock protein 70 has a net prosurvival effect. Heat shock protein 70 has both antiinflammatory and proinflammatory effects depending on the cell type, context, and intracellular or extracellular location. Intracellular effects are often antiinflammatory with inhibition of nuclear factor-kappaB signaling. Extracellular effects can lead to inflammatory cytokine production or induction of regulatory immune cells and reduced inflammation.
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Apoptose/fisiologia , Isquemia Encefálica/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Inflamação/etiologia , Animais , Morte Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Inflamação/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem. RESULTS: We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth. CONCLUSIONS: Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Padronização Corporal/genética , MicroRNAs/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Padronização Corporal/fisiologia , Perfilação da Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Fatores de Transcrição/genéticaRESUMO
Neutropenia is a common consequence of radiation and chemotherapy in cancer patients. The resulting immunocompromised patients become highly susceptible to potentially life-threatening infections. Granulocyte colony-stimulating factor (G-CSF) is known to stimulate neutrophil production and is widely used as a treatment of chemotherapy-induced neutropenia. A small-molecule G-CSF secretagogue without a requirement for refrigerated supply chain would offer a more convenient and cost-effective treatment of chemotherapy-induced neutropenia. Bacterial lipopeptides activate innate immune responses through Toll-like receptor 2 (TLR2) and induce the release of cytokines, including G-CSF, from macrophages, monocytes, and endothelial. Pam2CSK4 is a synthetic lipopeptide that effectively mimics bacterial lipoproteins known to activate TLR2 receptor signaling through the TLR2/6 heterodimer. Substrate-based drug design led to the discovery of GSK3277329, which stimulated the release of G-CSF in activated THP-1 cells, peripheral blood mononuclear cells, and human umbilical vein endothelial cells. When administered subcutaneously to cynomolgus monkeys (Macaca fascicularis), GSK3277329 caused systemic elevation of G-CSF and interleukin-6 (IL-6), but not IL-1ß or tumor necrosis factor α, indicating a selective cytokine-stimulation profile. Repeat daily injections of GSK3277329 in healthy monkeys also raised circulating neutrophils above the normal range over a 1-week treatment period. More importantly, repeated daily injections of GSK3277329 over a 2-week period restored neutrophil loss in monkeys given chemotherapy treatment (cyclophosphamide, Cytoxan). These data demonstrate preclinical in vivo proof of concept that TLR2 agonism can drive both G-CSF induction and subsequent neutrophil elevation in the cynomolgus monkey and could be a therapeutic strategy for the treatment of chemotherapy-induced neutropenia.
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The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.
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Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Quinoxalinas/farmacologia , Tanquirases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Quinoxalinas/síntese química , Quinoxalinas/química , Relação Estrutura-Atividade , Tanquirases/metabolismoRESUMO
CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.
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Caspases/genética , Caspases/metabolismo , Imunidade Inata , Ativação Linfocitária , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Animais , Linfócitos B/imunologia , Células Dendríticas/imunologia , Técnicas de Introdução de Genes , Inflamação/genética , Inflamação/imunologia , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação , Baço/imunologia , Linfócitos T/imunologiaRESUMO
NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.
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Benzotiazóis/farmacologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Xantinas/farmacologia , Animais , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Recent studies suggest a link between mitochondria and proinflammatory cytokine generation. We previously demonstrated that overexpression of mitochondrial chaperone glucose-regulated protein75 (Grp75/mortalin) protects mitochondria. In this study we investigated the modulation of the lipopolisaccharide (LPS)-induced inflammatory response of microglial BV-2 cells by Grp75. We demonstrate that LPS-induced activation promotes significant metabolic changes suppressing mitochondrial function and increasing glycolysis. Overexpression of Grp75 attenuates the LPS-induced oxidative and metabolic responses, and suppresses proinflammatory activation, which depends on both NF-κB activation and lactate. Thus overexpression of Grp75 provides a novel strategy to modulate proinflammatory cytokine production of relevance to inflammation-associated pathologies.
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Proteínas de Choque Térmico HSP70/genética , Proteínas de Membrana/genética , Microglia/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/genética , Animais , Células Cultivadas , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Proteínas de Choque Térmico HSP70/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Ácido Láctico/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Chaperonas Moleculares/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Plasmídeos , TransfecçãoRESUMO
NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.