MiR-155 Induction by Microbes/Microbial Ligands Requires NF-?B-Dependent de novo Protein Synthesis.

MiR-155 Induction by Microbes/Microbial Ligands Requires NF-?B-Dependent de novo Protein Synthesis.

Front Cell Infect Microbiol. 2012;2:73

Authors: Cremer TJ, Fatehchand K, Shah P, Gillette D, Patel H, Marsh RL, Besecker BY, Rajaram MV, Cormet-Boyaka E, Kanneganti TD, Schlesinger LS, Butchar JP, Tridandapani S

Abstract
MiR-155 regulates numerous aspects of innate and adaptive immune function. This miR is induced in response to Toll-like receptor ligands, cytokines, and microbial infection. We have previously shown that miR-155 is induced in monocytes/macrophages infected with Francisella tularensis and suppresses expression of the inositol phosphatase SHIP to enhance activation of the PI3K/Akt pathway, which in turn promotes favorable responses for the host. Here we examined how miR-155 expression is regulated during infection. First, our data demonstrate that miR-155 can be induced through soluble factors of bacterial origin and not the host. Second, miR-155 induction is not a direct effect of infection and it requires NF-?B signaling to up-regulate fos/jun transcription factors. Finally, we demonstrate that the requirement for NF-?B-dependent de novo protein synthesis is globally shared by microbial ligands and live bacteria. This study provides new insight into the complex regulation of miR-155 during microbial infection.

PMID: 22919664 [PubMed]

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Pertussis Toxin, an Inhibitor of G(?i) PCR, Inhibits Bile Acid- and Cytokine-Induced Apoptosis in Primary Rat Hepatocytes.

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Pertussis Toxin, an Inhibitor of G(?i) PCR, Inhibits Bile Acid- and Cytokine-Induced Apoptosis in Primary Rat Hepatocytes.

PLoS One. 2012;7(8):e43156

Authors: Karimian G, Buist-Homan M, Faber KN, Moshage H

Abstract
Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. G(?i)PCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting G(?i)PCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-? (TNF?)/actinomycin D (ActD). PT (50-200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNF?/ActD-induced apoptosis in rat hepatocytes (-60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. Conclusion: Pertussis toxin, an inhibitor of G(?i)PCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.

PMID: 22900098 [PubMed - in process]

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Engineered GPCRs as tools to modulate signal transduction.

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Engineered GPCRs as tools to modulate signal transduction.

Physiology (Bethesda). 2008 Dec;23:313-21

Authors: Pei Y, Rogan SC, Yan F, Roth BL

Abstract
Different families of G-protein-coupled receptors (GPCRs) have been engineered to provide exclusive control over the activation of these receptors and thus to understand better the consequences of their signaling in vitro and in vivo. These engineered receptors, named RASSLs (receptors activated solely by synthetic ligands) and DREADDs (designer receptors exclusively activated by designer drugs), are insensitive to their endogenous ligands but can be activated by synthetic drug-like compounds. Currently, the existing RASSLs and DREADDs cover the Gi, Gq, and Gs signaling pathways. These modified GPCRs can be utilized as ideal tools to study GPCR functions selectively in specific cellular populations.

PMID: 19074739 [PubMed - indexed for MEDLINE]

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Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

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Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

Biochemistry. 2004 Jun 29;43(25):8204-16

Authors: Waller A, Sutton KL, Kinzer-Ursem TL, Absood A, Traynor JR, Linderman JJ, Omann GM

Abstract
The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.

PMID: 15209517 [PubMed - indexed for MEDLINE]

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G-protein-coupled receptors in adult neurogenesis.

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G-protein-coupled receptors in adult neurogenesis.

Pharmacol Rev. 2012 Jul;64(3):645-75

Authors: Doze VA, Perez DM

Abstract
The importance of adult neurogenesis has only recently been accepted, resulting in a completely new field of investigation within stem cell biology. The regulation and functional significance of adult neurogenesis is currently an area of highly active research. G-protein-coupled receptors (GPCRs) have emerged as potential modulators of adult neurogenesis. GPCRs represent a class of proteins with significant clinical importance, because approximately 30% of all modern therapeutic treatments target these receptors. GPCRs bind to a large class of neurotransmitters and neuromodulators such as norepinephrine, dopamine, and serotonin. Besides their typical role in cellular communication, GPCRs are expressed on adult neural stem cells and their progenitors that relay specific signals to regulate the neurogenic process. This review summarizes the field of adult neurogenesis and its methods and specifies the roles of various GPCRs and their signal transduction pathways that are involved in the regulation of adult neural stem cells and their progenitors. Current evidence supporting adult neurogenesis as a model for self-repair in neuropathologic conditions, adult neural stem cell therapeutic strategies, and potential avenues for GPCR-based therapeutics are also discussed.

PMID: 22611178 [PubMed - in process]

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Subversion of innate immune responses by bacterial hindrance of NF-?B pathway.

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Subversion of innate immune responses by bacterial hindrance of NF-?B pathway.

Cell Microbiol. 2012 Feb;14(2):155-67

Authors: Le Negrate G

Abstract
Bacterial infections cause substantial mortality and burden of disease globally. Induction of a strong innate inflammatory response is the first common host mechanism required for elimination of the invading pathogens. The host transcription factor, nuclear factor kappa B (NF-?B) is essential for immune activation. Conversely, bacterial pathogens have evolved strategies to interfere directly with host cell signalling by regulating or mimicking host proteins. Given the key role of NF-?B in the host inflammatory response, bacteria have expectedly developed virulence effectors interfering with NF-?B signalling pathways. In this review, we explore the bacterial mechanisms utilized to prevent effective NF-?B signalling, which in turn usurp the host inflammatory response.

PMID: 22044780 [PubMed - indexed for MEDLINE]

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A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

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A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

Biochem Biophys Res Commun. 2011 Apr 1;407(1):18-22

Authors: Wu C, Du L, Wang D, Wang L, Zhao L, Wang P

Abstract
Olfactory receptors, which are responsible for sensing odor molecules, form the largest G protein-coupled receptor (GPCR) family in mammalian animals. These proteins play an important role in the detection of chemical signals and signal transduction to the brain. Currently, only a limited number of olfactory receptors have been characterized, which is mainly due to the lack of sensitive and efficient tools for performing functional assays of these receptors. This paper describes a novel surface acoustic wave (SAW)-based biosensor for highly sensitive functional assays of olfactory receptors. An olfactory receptor of Caenorhabditis elegans, ODR-10, was expressed on the plasma membrane of human breast cancer MCF-7 cells, which was used as a model system for this study. For specific odorant response assays, the membrane fraction of MCF-7 cells containing ODR-10 was extracted and integrated with our SAW sensors. The response of ODR-10 to various odorants was monitored by recording the resonance frequency shifts of SAWs applied to the sensor. Our results show that heterologously expressed ODR-10 receptors can specifically respond to diacetyl, its natural ligand. Dose-dependent responses were obtained by performing measurements using various concentrations of diacetyl. The sensitivity of this biosensor is 2kHz/ng and can detect concentrations as low as 10(-10)mM, which is 10� lower than what has previously been reported. This biosensor can be used to characterize odorant response profiles of olfactory receptors and provide information rich data for functional assays of olfactory receptors. In addition to providing a greater understanding of the biological mechanisms of GPCRs, such data holds great potential in many other fields such as food industry, biomedicine, and environmental protection.

PMID: 21333624 [PubMed - indexed for MEDLINE]

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Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-?B and MAPK activation, and acetylation of histone H3.

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Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-?B and MAPK activation, and acetylation of histone H3.

Arch Pharm Res. 2011 Dec;34(12):2109-16

Authors: Jeong JB, Hong SC, Jeong HJ, Koo JS

Abstract
Although inflammation acts as host defense mechanism against infection or injury and is primarily a self limiting process, inadequate resolution of inflammatory responses leads to various chronic disorders. This work aimed to elucidate the anti-inflammatory effects of 2-methoxy-4-vinylphenol (2M4VP) isolated from pine needles in LPS-stimulated RAW264.7 cells. Some key pro-inflammatory mediators including nitric oxide (NO), prostaglandins (PGE(2)), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied by sandwich ELISA and western blot. In addition, suppression of NF-?B and MAPK activation, and histone acetylation was studied by western blot analysis and immunostaining. 2M4VP dosedependently inhibited NO and PGE(2) production and also blocked LPS-induced iNOS and COX-2 expression. In addition, 2M4VP potently inhibited the translocation of NF-?B p65 into the nucleus by I?B degradation following I?B-? phosphorylation and the phosphorylation of MAPKs such as p38, ERK1/2, and JNK. Also, 2M4VP inhibited hyper-acetylation of histone H3 (Lys9/Lys14) induced by LPS. Taken together, our results suggest that 2M4VP, a naturally occurring phenolic compound, exert potent anti-inflammatory effects by inhibiting LPS-induced NO, PGE(2), iNOS, and COX-2 in RAW264.7 cells. These effects are mediated by suppression of NF-?B and MAPK activation and histone acetylation.

PMID: 22210037 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Classification of G-protein coupled receptors at four levels.

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Classification of G-protein coupled receptors at four levels.

Protein Eng Des Sel. 2006 Nov;19(11):511-6

Authors: Gao QB, Wang ZZ

Abstract
G-protein coupled receptors (GPCRs) are transmembrane proteins which via G-proteins initiate some of the important signaling pathways in a cell and are involved in various physiological processes. Thus, computational prediction and classification of GPCRs can supply significant information for the development of novel drugs in pharmaceutical industry. In this paper, a nearest neighbor method has been introduced to discriminate GPCRs from non-GPCRs and subsequently classify GPCRs at four levels on the basis of amino acid composition and dipeptide composition of proteins. Its performance is evaluated on a non-redundant dataset consisted of 1406 GPCRs for six families and 1406 globular proteins using the jackknife test. The present method based on amino acid composition achieved an overall accuracy of 96.4% and Matthew's correlation coefficient (MCC) of 0.930 for correctly picking out the GPCRs from globular proteins. The overall accuracy and MCC were further enhanced to 99.8% and 0.996 by dipeptide composition-based method. On the other hand, the present method has successfully classified 1406 GPCRs into six families with an overall accuracy of 89.6 and 98.8% using amino acid composition and dipeptide composition, respectively. For the subfamily prediction of 1181 GPCRs of rhodopsin-like family, the present method achieved an overall accuracy of 76.7 and 94.5% based on the amino acid composition and dipeptide composition, respectively. Finally, GPCRs belonging to the amine subfamily and olfactory subfamily of rhodopsin-like family were further analyzed at the type level. The overall accuracy of dipeptide composition-based method for the classification of amine type and olfactory type of GPCRs reached 94.5 and 86.9%, respectively, while the overall accuracy of amino acid composition-based method was very low for both subfamilies. In comparison with existing methods in the literature, the present method also displayed great competitiveness. These results demonstrate the effectiveness of our method on identifying and classifying GPCRs correctly. GPCRsIdentifier, a corresponding stand-alone executable program for GPCR identification and classification was also developed, which can be acquired freely on request from the authors for academic purposes.

PMID: 17032692 [PubMed - indexed for MEDLINE]

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CD40 stimulates a "feed-forward" NF-?B-driven molecular pathway that regulates IFN-? expression in carcinoma cells.

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CD40 stimulates a "feed-forward" NF-?B-driven molecular pathway that regulates IFN-? expression in carcinoma cells.

J Immunol. 2012 Jun 1;188(11):5521-7

Authors: Moschonas A, Ioannou M, Eliopoulos AG

Abstract
IFN-? and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses. In this study, we show that CD40 receptor occupancy results in IFN-? upregulation through an unconventional "feed-forward" mechanism, which is orchestrated by canonical NF-?B and involves the sequential de novo synthesis of IFN regulatory factor (IRF)1 and Viperin (RSAD2), an IRF1 target. RelA (p65) NF-?B, IRF1, and Viperin-dependent IRF7 binding to the IFN-? promoter largely controls its activity. However, full activation of IFN-? also requires the parallel engagement of noncanonical NF-?B2 signaling leading to p52 recruitment to the IFN-? promoter. These data define a novel link between CD40 signaling and IFN-? expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.

PMID: 22547704 [PubMed - indexed for MEDLINE]

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Cooperative roles of NF-?B and NFAT4 in polyomavirus JC regulation at the KB control element.

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Cooperative roles of NF-?B and NFAT4 in polyomavirus JC regulation at the KB control element.

Virology. 2012 Oct 10;432(1):146-54

Authors: Wollebo HS, Melis S, Khalili K, Safak M, White MK

Abstract
The human polyomavirus JC (JCV) is the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is extremely common and after primary infection, JCV persists in a latent state. However, PML is a very rare disease suggesting that the virus is tightly regulated. Previously, we showed that NF-?B and C/EBP? regulate the JCV early and late promoters via a DNA control element, KB, which also mediates the stimulatory effects of proinflammatory cytokines such as TNF-? on JCV gene expression. Other studies have implicated NFAT4 in JCV regulation. We now report that NFAT4 and NF-?B interact at the KB element to co-operatively activate both JCV early and late transcription and viral DNA replication. This interplay is inhibited by C/EBP? and by agents that block the calcineurin/NFAT signaling pathway. The importance of these events in the regulation of JCV latency and reactivation is discussed.

PMID: 22749879 [PubMed - in process]

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