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Sample records for inhibiting general protein

  1. Cardiac glycosides induce cell death in human cells by inhibiting general protein synthesis.

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    Andrea Perne

    2009-12-01

    Full Text Available Cardiac glycosides are Na(+/K(+-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive.Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+/K(+-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+/K(+-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels.The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.

  2. Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

    Science.gov (United States)

    Uluisik, Irem; Kaya, Alaattin; Fomenko, Dmitri E.; Karakaya, Huseyin C.; Carlson, Bradley A.; Gladyshev, Vadim N.; Koc, Ahmet

    2011-01-01

    Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance. PMID:22114689

  3. Boron stress activates the general amino acid control mechanism and inhibits protein synthesis.

    Directory of Open Access Journals (Sweden)

    Irem Uluisik

    Full Text Available Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.

  4. Generalization of fear inhibition by disrupting hippocampal protein synthesis-dependent reconsolidation process.

    Science.gov (United States)

    Yang, Chih-Hao; Huang, Chiung-Chun; Hsu, Kuei-Sen

    2011-09-01

    Repetitive replay of fear memories may precipitate the occurrence of post-traumatic stress disorder and other anxiety disorders. Hence, the suppression of fear memory retrieval may help prevent and treat these disorders. The formation of fear memories is often linked to multiple environmental cues and these interconnected cues may act as reminders for the recall of traumatic experiences. However, as a convenience, a simple paradigm of one cue pairing with the aversive stimulus is usually used in studies of fear conditioning in animals. Here, we built a more complex fear conditioning model by presenting several environmental stimuli during fear conditioning and characterize the effectiveness of extinction training and the disruption of reconsolidation process on the expression of learned fear responses. We demonstrate that extinction training with a single-paired cue resulted in cue-specific attenuation of fear responses but responses to other cures were unchanged. The cue-specific nature of the extinction persisted despite training sessions combined with D-cycloserine treatment reveals a significant weakness in extinction-based treatment. In contrast, the inhibition of the dorsal hippocampus (DH) but not the basolateral amygdala (BLA)-dependent memory reconsolidation process using either protein synthesis inhibitors or genetic disruption of cAMP-response-element-binding protein-mediated transcription comprehensively disrupted the learned connections between fear responses and all paired environmental cues. These findings emphasize the distinct role of the DH and the BLA in the reconsolidation process of fear memories and further indicate that the disruption of memory reconsolidation process in the DH may result in generalization of fear inhibition.

  5. NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

    International Nuclear Information System (INIS)

    Dodsworth, Jeremy A.; Leigh, John A.

    2007-01-01

    Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI 1,2 binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI 1,2 binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI 1,2 , decreasing its inhibitory effect. NifI 1,2 prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI 1,2 was unable to bind to an AlF 4 - -stabilized Fe protein:MoFe protein complex. NifI 1,2 inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI 1,2 competes with the Fe protein for binding to MoFe protein and prevents electron transfer

  6. Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

    Science.gov (United States)

    Fleishman, Sarel

    2012-02-01

    Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

  7. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Inhibition of Retinoblastoma Protein Inactivation

    Science.gov (United States)

    2017-11-01

    CONTRACT NUMBER Inhibition of Retinoblastoma Protein Inactivation 5b. GRANT NUMBER W81XWH-14-1-0329 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Seth M...confirmed 108 compounds as giving a dose-response curve with at least 30% inhibition at 10 µM. The flowchart of hit progression is shown on the...Cancer Research Program under Award No. W81XWH-14-1-0329 to S.M.R. Opinions, interpretations, conclusions, and recommendations are those of the author

  9. Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

    International Nuclear Information System (INIS)

    Minor, P.D.; Dimmock, N.J.

    1977-01-01

    Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

  10. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  11. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  12. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra

    DEFF Research Database (Denmark)

    Thomsen, S.; Hansen, Harald S.; Nyman, U.

    1991-01-01

    Phytolacca dodecandra (L'Herit) grown in cell cultures was investigated for content of ribosome-inhibiting proteins, which was evaluated hy measuring inhibition of protein synthesis in a cell-free rat liver extract. Calli initiated from leaf, cotyledon, radicle, and hypocotyl and suspension cells...

  13. Aedes aegypti D7 Saliva Protein Inhibits Dengue Virus Infection.

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    Michael J Conway

    2016-09-01

    Full Text Available Aedes aegypti is the primary vector of several medically relevant arboviruses including dengue virus (DENV types 1-4. Ae. aegypti transmits DENV by inoculating virus-infected saliva into host skin during probing and feeding. Ae. aegypti saliva contains over one hundred unique proteins and these proteins have diverse functions, including facilitating blood feeding. Previously, we showed that Ae. aegypti salivary gland extracts (SGEs enhanced dissemination of DENV to draining lymph nodes. In contrast, HPLC-fractionation revealed that some SGE components inhibited infection. Here, we show that D7 proteins are enriched in HPLC fractions that are inhibitory to DENV infection, and that recombinant D7 protein can inhibit DENV infection in vitro and in vivo. Further, binding assays indicate that D7 protein can directly interact with DENV virions and recombinant DENV envelope protein. These data reveal a novel role for D7 proteins, which inhibits arbovirus transmission to vertebrates through a direct interaction with virions.

  14. Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations.

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    Sinjan Choudhary

    Full Text Available We report here interesting synergistic effects of proline and sorbitol, two well-known chemical chaperones, in the inhibition of fibrillation of two proteins, insulin and lysozyme. A combination of many biophysical techniques has been used to understand the structural morphology and modes of interaction of the chaperones with the proteins during fibrillation. Both the chaperones establish stronger polar interactions in the elongation and saturation stages of fibrillation compared to that in the native stage. However, when presented as a mixture, we also see contribution of hydrophobic interactions. Thus, a co-operative adjustment of polar and hydrophobic interactions between the chaperones and the protein surface seems to drive the synergistic effects in the fibrillation process. In insulin, this synergy is quantitatively similar in all the stages of the fibrillation process. These observations would have significant implications for understanding protein folding concepts, in general, and for designing combination therapies against protein fibrillation, in particular.

  15. Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations.

    Science.gov (United States)

    Choudhary, Sinjan; Save, Shreyada N; Kishore, Nand; Hosur, Ramakrishna V

    2016-01-01

    We report here interesting synergistic effects of proline and sorbitol, two well-known chemical chaperones, in the inhibition of fibrillation of two proteins, insulin and lysozyme. A combination of many biophysical techniques has been used to understand the structural morphology and modes of interaction of the chaperones with the proteins during fibrillation. Both the chaperones establish stronger polar interactions in the elongation and saturation stages of fibrillation compared to that in the native stage. However, when presented as a mixture, we also see contribution of hydrophobic interactions. Thus, a co-operative adjustment of polar and hydrophobic interactions between the chaperones and the protein surface seems to drive the synergistic effects in the fibrillation process. In insulin, this synergy is quantitatively similar in all the stages of the fibrillation process. These observations would have significant implications for understanding protein folding concepts, in general, and for designing combination therapies against protein fibrillation, in particular.

  16. Inhibiting Translation One Protein at a Time.

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    Disney, Matthew D

    2017-06-01

    Historically, translational inhibitors have been confined to anti-bacterials that globally affect translation. Lintner et al. demonstrate that small molecules can specifically inhibit translation of a single disease-associated protein by stalling the ribosome's nascent chain [1], opening up a new therapeutic strategy for 'undruggable' proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. General anesthetics inhibit erythropoietin induction under hypoxic conditions in the mouse brain.

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    Tomoharu Tanaka

    Full Text Available Erythropoietin (EPO, originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS. EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes.BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10-1.0%. Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA.Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.

  18. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  19. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma

    NARCIS (Netherlands)

    Elisen, M. G.; von dem Borne, P. A.; Bouma, B. N.; Meijers, J. C.

    1998-01-01

    Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited

  20. The C protein of measles virus inhibits the type I interferon response

    International Nuclear Information System (INIS)

    Shaffer, Jessica A.; Bellini, William J.; Rota, Paul A.

    2003-01-01

    Type I interferons (IFNα/β) are an important part of innate immunity to viral infections because they induce an antiviral response and limit viral replication until the adaptive response clears the infection. Since the nonstructural proteins of several paramyxoviruses inhibit the IFNα/β response, we chose to explore the role of the C protein of measles virus (MV) in such inhibition. Previous studies have suggested that the MV C protein may serve as a virulence factor, but its role in the pathogenesis of MV remains undefined. In the present study, a recombinant MV strain that does not express the C protein (MV C-) and its parental strain (Ed Tag) were used. Growth of MV C- was restricted in human peripheral blood mononuclear cells and HeLa cells, but in the presence of neutralizing antibodies to IFNα/β, MV C- produced titers that were equivalent to those of Ed Tag. In addition, expression of the MV C protein from plasmid DNA inhibited the production of an IFNα/β responsive reporter gene and, to a lesser extent, inhibited an IFNγ responsive reporter gene. The ability of the MV C protein to suppress the IFNα/β response was confirmed using a biologic assay. After IFNβ stimulation, HeLa cells infected with Ed Tag produced five-fold less IFNα/β than cells infected with MV C-. While the mechanism of inhibition remains unclear, these data suggest that the MV C protein plays an important role in the pathogenesis of MV by inhibiting IFNα/β signaling

  1. General protein-protein cross-linking.

    Science.gov (United States)

    Alegria-Schaffer, Alice

    2014-01-01

    This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). © 2014 Elsevier Inc. All rights reserved.

  2. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    Energy Technology Data Exchange (ETDEWEB)

    Mena, Natalia P. [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Bulteau, Anne Laure [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Salazar, Julio [Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Hirsch, Etienne C. [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Nunez, Marco T., E-mail: mnunez@uchile.cl [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile)

    2011-06-03

    Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

  3. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    International Nuclear Information System (INIS)

    Mena, Natalia P.; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C.; Nunez, Marco T.

    2011-01-01

    Highlights: → Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. → Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. → Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. → Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex

  4. Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

    International Nuclear Information System (INIS)

    Brooks, J.C.; Brooks, M.

    1985-01-01

    Permeabilized cells treated with the adenosine triphosphate analog, ( 35 S)adenosine-5'-0-3(3-thiotriphosphate) ((γ- 35 S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35 S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35 S from the cells over a 20 min period. Treatment of permeabilized cells with ATPγS inhibited secretion and 35 S incorporation into the cells. Pretreatment with ATPγS resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35 S from (γ- 35 S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables

  5. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

    Science.gov (United States)

    Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

    2015-10-01

    The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

  6. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  7. Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin

    International Nuclear Information System (INIS)

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A.

    1987-01-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na + by atrial natriuretic peptide and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK i . Using 22 Na + fluxes, they further investigated the modulation of Na + transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na + uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na + uptake by 93 ± 13 and 51 ± 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK i cells, inhibits 22 Na + influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na + uptake. These events may be sequentially involved in the action of atrial natriuretic peptide

  8. Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic

  9. Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

    Science.gov (United States)

    Kleihues, P.; Magee, P. N.

    1973-01-01

    1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2–3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[14C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA. PMID:4774397

  10. Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

  11. Inhibition of Nonenzymatic Protein Glycation by Pomegranate and Other Fruit Juices

    Science.gov (United States)

    Dorsey, Pamela Garner; Greenspan, Phillip

    2014-01-01

    Abstract The nonenzymatic glycation of proteins and the formation of advanced glycation endproducts in diabetes leads to the crosslinking of proteins and disease complications. Our study sought to demonstrate the effect of commonly consumed juices (pomegranate, cranberry, black cherry, pineapple, apple, and Concord grape) on the fructose-mediated glycation of albumin. Albumin glycation decreased by 98% in the presence of 10 μL of pomegranate juice/mL; other juices inhibited glycation by only 20%. Pomegranate juice produced the greatest inhibition on protein glycation when incubated at both the same phenolic concentration and the same antioxidant potential. Both punicalagin and ellagic acid significantly inhibited the glycation of albumin by ∼90% at 5 μg/mL. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that pomegranate, but not apple juice, protected albumin from modification. These results demonstrate that pomegranate juice and two of its major constituents are potent inhibitors of fructose-mediated protein glycation. PMID:24433074

  12. Cholesteryl ester transfer protein (cetp) inhibition in the treatment of cancer

    KAUST Repository

    Kaur, Mandeep; Esau, Luke E.; Sagar, Sunii

    2016-01-01

    In one embodiment, the invention provides methods of treatment which use therapeutically effective amounts of Choleste ryl Ester Transfer Protein (CETP) inhibitors to treat a variety of cancers. In certain embodiments, the inhibitor is a CETP-inhibiting small molecule, CETP-inhibiting antisense oligonucleotide, CETP-inhibiting siRNA or a CETP- inhibiting antibody. Related pharmaceutical compositions, kits, diagnostics and screens are also provided.

  13. Cholesteryl ester transfer protein (cetp) inhibition in the treatment of cancer

    KAUST Repository

    Kaur, Mandeep

    2016-09-01

    In one embodiment, the invention provides methods of treatment which use therapeutically effective amounts of Choleste ryl Ester Transfer Protein (CETP) inhibitors to treat a variety of cancers. In certain embodiments, the inhibitor is a CETP-inhibiting small molecule, CETP-inhibiting antisense oligonucleotide, CETP-inhibiting siRNA or a CETP- inhibiting antibody. Related pharmaceutical compositions, kits, diagnostics and screens are also provided.

  14. DCB-3503, a tylophorine analog, inhibits protein synthesis through a novel mechanism.

    Directory of Open Access Journals (Sweden)

    Ying Wang

    Full Text Available BACKGROUND: DCB-3503, a tylophorine analog, inhibits the growth of PANC-1 (human pancreatic ductal cancer cell line and HepG2 (human hepatocellular cancer cell line tumor xenografts in nude mice. The inhibition of growth leads to cancer cell differentiation instead of cell death. However, the mechanisms of action of tylophorine analogs is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that DCB-3503 suppresses the expression of pro-oncogenic or pro-survival proteins with short half-lives, including cyclin D1, survivin, beta-catenin, p53, and p21, without decreasing their mRNA levels. Proteasome inhibitor reversed the inhibitory effect of DCB-3503 on expression of these proteins. DCB-3503 inhibited the incorporation of radiolabeled amino acid and thymidine, and to a much lesser degree of uridine, in a panel of cell lines. The mechanism of inhibition of protein synthesis is different from that of cycloheximide (CHX as assayed in cell culture and HeLa in vitro translation system. Furthermore, in contrast to rapamycin, DCB-3503 does not affect protein synthesis through the mTOR pathway. DCB-3503 treatment shifts the sedimentation profiles of ribosomes and mRNAs towards the polysomal fractions while diminishing monosome abundance, indicative of the inhibition of the elongation step of protein synthesis. Preferential down regulation of several studied proteins under these conditions is likely due to the relative short half-lives of these proteins. CONCLUSION/SIGNIFICANCE: The inhibitory effect of DCB-3503 on translation is apparently distinct from any of the current anticancer compounds targeting protein synthesis. Translation inhibitors with novel mechanism could complement current chemotherapeutic agents for the treatment of human cancers and suppress the occurrence of drug resistance.

  15. Inhibition of chloroplast protein synthesis following light chilling of tomato

    International Nuclear Information System (INIS)

    Kent, J.; Ort, D.

    1989-01-01

    In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

  16. Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

    Science.gov (United States)

    Rabbani, M A G; Ribaudo, Michael; Guo, Ju-Tao; Barik, Sailen

    2016-12-15

    A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-β. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3. Copyright © 2016, American Society for Microbiology

  17. The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

    International Nuclear Information System (INIS)

    Cross, Janet V; Foss, Frank W; Rady, Joshua M; Macdonald, Timothy L; Templeton, Dennis J

    2007-01-01

    Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of 'Phase 2' enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. These results demonstrate that MEKK1 is directly modified and inhibited by

  18. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  19. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  20. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    International Nuclear Information System (INIS)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

    2015-01-01

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

  1. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

  2. Tubule urate and PAH transport: sensitivity and specificity of serum protein inhibition

    International Nuclear Information System (INIS)

    Grantham, J.J.; Kennedy, J.; Cowley, B.

    1987-01-01

    Macromolecules in rabbit serum inhibit the cellular uptake and transepithelial secretion of [ 14 C]urate and p-[ 3 H]aminohippurate ([ 3 H]PAH) in rabbit S 2 proximal tubule segments. To understand better the potential role these inhibitors may have in the regulation of renal organic anion excretion, the authors examined the specificity and relative inhibitory effects on tubule urate and PAH transport of albumin and γ-globulin, the major inhibitory proteins in rabbit serum. Native rabbit serum markedly inhibited the cellular accumulation or urate and PAH by isolated nonperfused segments. Urate and PAH transport was also inhibited by bovine serum, human serum, Cohn-fractionated rabbit albumin, and rabbit γ-globulin, but not by Cohn-fractionated bovine serum albumin. α-Lactalbumin and β-lactoglobulin, derived from milk, also inhibited urate and PAH transport, but to a lesser extent than albumin and γ-globulin. The transport inhibitory effects of proteins were independent of their binding to urate and PAH. Unidirectional influx and the steady-state intracellular accumulation of urate and PAH in suspensions of proximal tubules were decreased by rabbit serum proteins, suggesting that these inhibitors act on the external face of the cells to diminish the uptake of the organic anions. These studies indicate that the principal plasma proteins (albumin and γ-globulin) significantly inhibit urate and PAH transporters in the basolateral membranes of S 2 proximal tubules. They suggest that circulating plasma proteins that can penetrate the basement membrane of proximal tubules may directly modulate the renal excretion of urate and PAH

  3. Recombinant human prion protein inhibits prion propagation in vitro.

    Science.gov (United States)

    Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

    2013-10-09

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

  4. Maribavir Inhibits Epstein-Barr Virus Transcription through the EBV Protein Kinase

    Science.gov (United States)

    Whitehurst, Christopher B.; Sanders, Marcia K.; Law, Mankit; Wang, Fu-Zhang; Xiong, Jie; Dittmer, Dirk P.

    2013-01-01

    Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus. PMID:23449792

  5. Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

    International Nuclear Information System (INIS)

    Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

    2003-01-01

    The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

  6. The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

    Directory of Open Access Journals (Sweden)

    Macdonald Timothy L

    2007-09-01

    Full Text Available Abstract Background Dietary isothiocyanates (ITCs are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results

  7. Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

    International Nuclear Information System (INIS)

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity [ 3 H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 1 -antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [ 3 H]-imipramine binding site

  8. Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

    Directory of Open Access Journals (Sweden)

    Richa Tyagi

    2015-02-01

    Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

  9. Antiviral Protein of Momordica charantia L. Inhibits Different Subtypes of Influenza A

    Directory of Open Access Journals (Sweden)

    Viroj Pongthanapisith

    2013-01-01

    Full Text Available The new antiviral activity of the protein extracted from Momordica charantia was determined with different subtypes of influenza A. The protein was purified from the seed of M. charantia using an anion exchanger and a Fast Protein Liquid Chromatography (FPLC system. At the concentration of 1.401 mg/mL, the protein did not exhibit cytotoxicity in Madin-Darby canine kidney cells (MDCK but inhibited FFU influenza A/PR/8/34 H1N1 virus at 56.50%, 65.72%, and 100% inhibition by the protein treated before the virus (pretreated, the protein treated alongside with the virus (simultaneously treated, and the protein treated after the virus (posttreated during incubation, respectively. Using 5, 25, and 100 TCID50 of influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2 and A/Thailand/1(KAN-1/2004 H5N1, the IC50 was calculated to be 100, 150, and 200; 75, 175, and 300; and 40, 75, and 200 μg/mL, respectively. Our present finding indicated that the plant protein inhibited not only H1N1 and H3N2 but also H5N1 subtype. As a result of the broad spectrum of its antiviral activity, this edible plant can be developed as an effective therapeutic agent against various and even new emerging subtypes of influenza A.

  10. Effect of Glucuronidation on the Potential of Kaempferol to Inhibit Serine/Threonine Protein Kinases

    NARCIS (Netherlands)

    Beekmann, Karsten; Haan, De Laura H.J.; Actis-Goretta, Lucas; Bladeren, Van Peter J.; Rietjens, Ivonne M.C.M.

    2016-01-01

    To study the effect of metabolic conjugation of flavonoids on the potential to inhibit protein kinase activity, the inhibitory effects of the dietary flavonol kaempferol and its major plasma conjugate kaempferol-3-O-glucuronide on protein kinases were studied. To this end, the inhibition of the

  11. The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

    Directory of Open Access Journals (Sweden)

    Chenjing Shang

    Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

  12. Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

    Science.gov (United States)

    Hanas, J S; Rodgers, J S; Bantle, J A; Cheng, Y G

    1999-11-01

    The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

  13. A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

  14. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  15. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    Science.gov (United States)

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

  16. Growth inhibition and differences in protein profiles in azadirachtin-treated Drosophila melanogaster larvae.

    Science.gov (United States)

    Wang, Hao; Lai, Duo; Yuan, Mei; Xu, Hanhong

    2014-04-01

    Azadirachtin A is a very effective biopesticide widely used in insect pest control. It has strong antifeeding and growth inhibitory activity against most insects, however, its mode of action is still unclear. Proteomic experiments using 2DE indicate significant effects of Azadirachtin A on the amount of proteins related to growth inhibition in Drosophila melanogaster larvae. Twenty-one spots with different intensity in azadirachtin-treated larvae were identified. These proteins are involved in cytoskeletal organization, transcription and translation, hormonal regulation, and energy metabolism. Protein network analysis reveals heat shock protein 23 to be a potential target of azadirachtin. These results provide new insights into understanding the mechanism of growth inhibition in insects in response to azadirachtin. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

    Science.gov (United States)

    Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

    2018-04-03

    The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

  18. Protein disulfide isomerase interacts with tau protein and inhibits its fibrillization.

    Directory of Open Access Journals (Sweden)

    Li-Rong Xu

    Full Text Available BACKGROUND: Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization. METHODOLOGY/PRINCIPAL FINDINGS: As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244-372 monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244-372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244-372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244-372 fibrillization more strongly than full-length human PDI. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau

  19. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  20. Zinc-finger antiviral protein inhibits XMRV infection.

    Directory of Open Access Journals (Sweden)

    Xinlu Wang

    Full Text Available BACKGROUND: The zinc-finger antiviral protein (ZAP is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV, HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV is a recently identified γ-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

  1. Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key.

    Directory of Open Access Journals (Sweden)

    Utpal Das

    Full Text Available BACKGROUND: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. METHODOLOGY: We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Abeta(1-42 peptide is modified. Changes in the hydrodynamic volume of Abeta(1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Abeta(1-42. Arginine increases the solubility of Abeta(1-42 peptide in aqueous medium. It decreases the aggregation of Abeta(1-42 as observed by atomic force microscopy. CONCLUSIONS: Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

  2. The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

    Science.gov (United States)

    Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

    2014-05-20

    Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for

  3. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  4. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition.

    Science.gov (United States)

    Moll, Lorna; Ben-Gedalya, Tziona; Reuveni, Hadas; Cohen, Ehud

    2016-04-01

    The discovery that the alteration of aging by reducing the activity of the insulin/IGF-1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration-linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF-1 signaling inhibitor, protects model worms from the aggregation of amyloid β peptide and polyglutamine peptides that are linked to the manifestation of Alzheimer's and Huntington's diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as "aggresomes." NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF-1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.-Moll, L., Ben-Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition. © FASEB.

  5. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.

    Science.gov (United States)

    Zhou, Tao; Dang, Ying; Zheng, Yong-Hui

    2014-03-01

    The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins

  6. Aptamer-Conjugated Calcium Phosphate Nanoparticles for Reducing Diabetes Risk via Retinol Binding Protein 4 Inhibition.

    Science.gov (United States)

    Torabi, Raheleh; Ghourchian, Hedayatollah; Amanlou, Massoud; Pasalar, Parvin

    2017-06-01

    Inhibition of the binding of retinol to its carrier, retinol binding protein 4, is a new strategy for treating type 2 diabetes; for this purpose, we have provided an aptamer-functionalized multishell calcium phosphate nanoparticle. First, calcium phosphate nanoparticles were synthesized and conjugated to the aptamer. The cytotoxicity of nanoparticles releases the process of aptamer from nanoparticles and their inhibition function of binding retinol to retinol binding protein 4. After synthesizing and characterizing the multishell calcium phosphate nanoparticles and observing the noncytotoxicity of conjugate, the optimum time (48 hours) and the pH (7.4) for releasing the aptamer from the nanoparticles was determined. The half-maximum inhibitory concentration (IC 50 ) value for inhibition of retinol binding to retinol binding protein 4 was 210 femtomolar (fmol). The results revealed that the aptamer could prevent connection between retinol and retinol binding protein 4 at a very low IC 50 value (210 fmol) compared to other reported inhibitors. It seems that this aptamer could be used as an efficient candidate not only for decreasing the insulin resistance in type 2 diabetes, but also for inhibiting the other retinol binding protein 4-related diseases. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

  7. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    International Nuclear Information System (INIS)

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory

    2007-01-01

    Frog virus 3 (FV3) is a large DNA virus that encodes ∼ 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIα). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIα triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins

  8. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  9. Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Mörck, Catarina; Elmelund-Præstekær, Louise Cathrine Braun; Kurth, Caroline

    2009-01-01

    of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C....... elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER...... and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C...

  10. Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

    Energy Technology Data Exchange (ETDEWEB)

    Ciomperlik, Jessica J. [Institute for Molecular Virology, and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI (United States); Basta, Holly A. [Department of Biology, Rocky Mountain College, Billings, MT (United States); Palmenberg, Ann C., E-mail: acpalmen@wisc.edu [Institute for Molecular Virology, and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI (United States)

    2015-10-15

    Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases.

  11. Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

    International Nuclear Information System (INIS)

    Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

    2015-01-01

    Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases

  12. A Mini Zinc-Finger Protein (MIF from Gerbera hybrida Activates the GASA Protein Family Gene, GEG, to Inhibit Ray Petal Elongation

    Directory of Open Access Journals (Sweden)

    Meixiang Han

    2017-09-01

    Full Text Available Petal appearance is an important horticultural trail that is generally used to evaluate the ornamental value of plants. However, knowledge of the molecular regulation of petal growth is mostly derived from analyses of Arabidopsis thaliana, and relatively little is known about this process in ornamental plants. Previously, GEG (Gerbera hybrida homolog of the gibberellin [GA]–stimulated transcript 1 [GAST1] from tomato, a gene from the GA stimulated Arabidopsis (GASA family, was reported to be an inhibitor of ray petal growth in the ornamental species, G. hybrida. To explore the molecular regulatory mechanism of GEG in petal growth inhibition, a mini zinc-finger protein (MIF was identified using yeast one-hybrid (Y1H screen. The direct binding of GhMIF to the GEG promoter was verified by using an electrophoretic mobility shift assay and a dual-luciferase assay. A yeast two-hybrid (Y2H revealed that GhMIF acts as a transcriptional activator. Transient transformation assay indicated that GhMIF is involved in inhibiting ray petal elongation by activating the expression of GEG. Spatiotemporal expression analyses and hormone treatment assay showed that the expression of GhMIF and GEG is coordinated during petal development. Taken together, these results suggest that GhMIF acts as a direct transcriptional activator of GEG, a gene from the GASA protein family to regulate the petal elongation.

  13. Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    2009-06-01

    Full Text Available Raf Kinase Inhibitory Protein (RKIP, also PEBP1, a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/- mouse embryonic fibroblasts (MEFs to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/- MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

  14. Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

    Science.gov (United States)

    Shemon, Anne N; Eves, Eva M; Clark, Matthew C; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira; Koide, Shohei; Rosner, Marsha Rich

    2009-06-24

    Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

  15. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    Directory of Open Access Journals (Sweden)

    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  16. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

    2015-10-23

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases

  17. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

    International Nuclear Information System (INIS)

    Tan, Yongsheng; Li, Yan

    2015-01-01

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 "l"o"w and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96"®Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 "l"o"w, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases expression of NR4A1

  18. Inhibition of protein kinase C induces differentiation in Neuro-2a cells

    International Nuclear Information System (INIS)

    Minana, M.D.; Felipo, V.; Grisolia, S.

    1990-01-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

  19. Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

    Science.gov (United States)

    Browning, Cynthia L; Wise, John Pierce

    2017-09-15

    Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

    Science.gov (United States)

    Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

    2008-01-01

    Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

  1. Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

    International Nuclear Information System (INIS)

    Maruyama, I.; Majerus, P.W.

    1987-01-01

    We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of 125 I-thrombin-thrombomodulin complexes, but not 125 I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of 125 I-thrombin and diisopropylphosphoryl (DIP) 125 I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C

  2. D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of); Baek, Won-Ki, E-mail: wonki@dsmc.or.kr [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of)

    2009-04-24

    D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

  3. Inhibition of hydrogenase synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum

    International Nuclear Information System (INIS)

    Novak, P.D.; Maier, R.J.

    1987-01-01

    Derepression of an uptake hydrogenase in Bradyrhizobium japonicum is dependent on a microaerophilic environment. Addition of DNA gyrase inhibitors during derepression of hydrogenase specifically prevented expression of the hydrogenase enzyme. Antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. The general pattern of proteins synthesized from 14 C-labeled amino acids during derepression was no significantly different whether proteins were labeled in the presence or in the absence of gyrase inhibitors. In contrast, if transcription or translation was inhibited by addition of inhibitors of those functions, virtually no proteins were labeled during derepression. This indicated that most of the 14 C-labeled proteins were synthesized de novo during derepression, synthesis of most proteins was unaffected by gyrase inhibitors, and the dependence of hydrogenase synthesis on gyrase activity was a specific one

  4. Curcumin ameliorates skeletal muscle atrophy in type 1 diabetic mice by inhibiting protein ubiquitination.

    Science.gov (United States)

    Ono, Taisuke; Takada, Shingo; Kinugawa, Shintaro; Tsutsui, Hiroyuki

    2015-09-01

    What is the central question of this study? We sought to examine whether curcumin could ameliorate skeletal muscle atrophy in diabetic mice by inhibiting protein ubiquitination, inflammatory cytokines and oxidative stress. What is the main finding and its importance? We found that curcumin ameliorated skeletal muscle atrophy in streptozotocin-induced diabetic mice by inhibiting protein ubiquitination without affecting protein synthesis. This favourable effect of curcumin was possibly due to the inhibition of inflammatory cytokines and oxidative stress. Curcumin may be beneficial for the treatment of muscle atrophy in type 1 diabetes mellitus. Skeletal muscle atrophy develops in patients with diabetes mellitus (DM), especially in type 1 DM, which is associated with chronic inflammation. Curcumin, the active ingredient of turmeric, has various biological actions, including anti-inflammatory and antioxidant properties. We hypothesized that curcumin could ameliorate skeletal muscle atrophy in mice with streptozotocin-induced type 1 DM. C57BL/6 J mice were injected with streptozotocin (200 mg kg(-1) i.p.; DM group) or vehicle (control group). Each group of mice was randomly subdivided into two groups of 10 mice each and fed a diet with or without curcumin (1500 mg kg(-1) day(-1)) for 2 weeks. There were significant decreases in body weight, skeletal muscle weight and cellular cross-sectional area of the skeletal muscle in DM mice compared with control mice, and these changes were significantly attenuated in DM+Curcumin mice without affecting plasma glucose and insulin concentrations. Ubiquitination of protein was increased in skeletal muscle from DM mice and decreased in DM+Curcumin mice. Gene expressions of muscle-specific ubiquitin E3 ligase atrogin-1/MAFbx and MuRF1 were increased in DM and inhibited in DM+Curcumin mice. Moreover, nuclear factor-κB activation, concentrations of the inflammatory cytokines tumour necrosis factor-α and interleukin-1β and oxidative

  5. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    International Nuclear Information System (INIS)

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

    2005-01-01

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

  6. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    OpenAIRE

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  7. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

    Science.gov (United States)

    Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

  8. A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH-stimulated luteinizing hormone (LH secretion

    Directory of Open Access Journals (Sweden)

    Musgrove Lois C

    2001-11-01

    Full Text Available Abstract Background Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs. These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. Results A truncated version of RGS3 (RGS3T = RGS3 314–519 inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein. Conclusions RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino

  9. Potent and Selective Peptide-based Inhibition of the G Protein Gαq*

    Science.gov (United States)

    Charpentier, Thomas H.; Waldo, Gary L.; Lowery-Gionta, Emily G.; Krajewski, Krzysztof; Strahl, Brian D.; Kash, Thomas L.; Harden, T. Kendall; Sondek, John

    2016-01-01

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells. PMID:27742837

  10. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  11. Interaction of technetium and molybdenum in nitrogenase inhibition in Azotobacter chroococcum

    International Nuclear Information System (INIS)

    Vandecasteele, C.M.; Henrot, J.; Van Hove, C.; Myttenaere, C.; Cogneau, M.

    1986-01-01

    Azotobacter chroococcum cultures were grown with and without Tc, using 99 Mo and sup(95m)Tc as Mo and Tc tracers. Tc does inhibit nitrogenase and Mo accumulation, but does not affect the Mo content of nitrogenase MoFe protein and seems to have less affinity for this than for protein compounds in general. Hence the inhibition is unlikely to be due to substitution of Mo by Tc. (UK)

  12. Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine.

    Science.gov (United States)

    McFarlane, Matthew R; Liang, Guosheng; Engelking, Luke J

    2014-01-24

    Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

  13. Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine*

    Science.gov (United States)

    McFarlane, Matthew R.; Liang, Guosheng; Engelking, Luke J.

    2014-01-01

    Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes. PMID:24337570

  14. BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

    Science.gov (United States)

    Cekanova, Maria; Fernando, Romaine I; Siriwardhana, Nalin; Sukhthankar, Mugdha; De la Parra, Columba; Woraratphoka, Jirayus; Malone, Christine; Ström, Anders; Baek, Seung J; Wade, Paul A; Saxton, Arnold M; Donnell, Robert M; Pestell, Richard G; Dharmawardhane, Suranganie; Wimalasena, Jay

    2015-02-01

    We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  16. Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

    Science.gov (United States)

    Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

    2016-12-02

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Betulinic acid selectively increases protein degradation and enhances prostate cancer-specific apoptosis: possible role for inhibition of deubiquitinase activity.

    Directory of Open Access Journals (Sweden)

    Teresita Reiner

    Full Text Available Inhibition of the ubiquitin-proteasome system (UPS of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs, which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy.

  18. Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

    Science.gov (United States)

    Al-Ramahi, Ismael; Panapakkam Giridharan, Sai Srinivas; Chen, Yu-Chi; Patnaik, Samarjit; Safren, Nathaniel; Hasegawa, Junya; de Haro, Maria; Wagner Gee, Amanda K; Titus, Steven A; Jeong, Hyunkyung; Clarke, Jonathan; Krainc, Dimitri; Zheng, Wei; Irvine, Robin F; Barmada, Sami; Ferrer, Marc; Southall, Noel; Weisman, Lois S; Botas, Juan; Marugan, Juan Jose

    2017-12-26

    The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

  19. Annotating activation/inhibition relationships to protein-protein interactions using gene ontology relations.

    Science.gov (United States)

    Yim, Soorin; Yu, Hasun; Jang, Dongjin; Lee, Doheon

    2018-04-11

    Signaling pathways can be reconstructed by identifying 'effect types' (i.e. activation/inhibition) of protein-protein interactions (PPIs). Effect types are composed of 'directions' (i.e. upstream/downstream) and 'signs' (i.e. positive/negative), thereby requiring directions as well as signs of PPIs to predict signaling events from PPI networks. Here, we propose a computational method for systemically annotating effect types to PPIs using relations between functional information of proteins. We used regulates, positively regulates, and negatively regulates relations in Gene Ontology (GO) to predict directions and signs of PPIs. These relations indicate both directions and signs between GO terms so that we can project directions and signs between relevant GO terms to PPIs. Independent test results showed that our method is effective for predicting both directions and signs of PPIs. Moreover, our method outperformed a previous GO-based method that did not consider the relations between GO terms. We annotated effect types to human PPIs and validated several highly confident effect types against literature. The annotated human PPIs are available in Additional file 2 to aid signaling pathway reconstruction and network biology research. We annotated effect types to PPIs by using regulates, positively regulates, and negatively regulates relations in GO. We demonstrated that those relations are effective for predicting not only signs, but also directions of PPIs. The usefulness of those relations suggests their potential applications to other types of interactions such as protein-DNA interactions.

  20. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

    Science.gov (United States)

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-07-13

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

  1. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

    Science.gov (United States)

    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  2. Soy protein isolate inhibits hepatic tumor promotion in mice fed a high-fat liquid diet.

    Science.gov (United States)

    Mercer, Kelly E; Pulliam, Casey F; Pedersen, Kim B; Hennings, Leah; Ronis, Martin Jj

    2017-03-01

    Alcoholic and nonalcoholic fatty liver diseases are risk factors for development of hepatocellular carcinoma, but the underlying mechanisms are poorly understood. On the other hand, ingestion of soy-containing diets may oppose the development of certain cancers. We previously reported that replacing casein with a soy protein isolate reduced tumor promotion in the livers of mice with alcoholic liver disease after feeding a high fat ethanol liquid diet following initiation with diethylnitrosamine. Feeding soy protein isolate inhibited processes that may contribute to tumor promotion including inflammation, sphingolipid signaling, and Wnt/β-catenin signaling. We have extended these studies to characterize liver tumor promotion in a model of nonalcoholic fatty liver disease produced by chronic feeding of high-fat liquid diets in the absence of ethanol. Mice treated with diethylnitrosamine on postnatal day 14 were fed a high-fat liquid diet made with casein or SPI as the sole protein source for 16 weeks in adulthood. Relative to mice fed normal chow, a high fat/casein diet led to increased tumor promotion, hepatocyte proliferation, steatosis, and inflammation. Replacing casein with soy protein isolate counteracted these effects. The high fat diets also resulted in a general increase in transcripts for Wnt/β-catenin pathway components, which may be an important mechanism, whereby hepatic tumorigenesis is promoted. However, soy protein isolate did not block Wnt signaling in this nonalcoholic fatty liver disease model. We conclude that replacing casein with soy protein isolate blocks development of steatosis, inflammation, and tumor promotion in diethylnitrosamine-treated mice fed high fat diets. Impact statement The impact of dietary components on cancer is a topic of great interest for both the general public and the scientific community. Liver cancer is currently the second leading form of cancer deaths worldwide. Our study has addressed the effect of the protein

  3. BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion

    Energy Technology Data Exchange (ETDEWEB)

    Cekanova, Maria, E-mail: mcekanov@utk.edu [Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Fernando, Romaine I. [Department of Obstetrics and Gynecology, Graduate School of Medicine, Medical Center, The University of Tennessee, Knoxville, TN (United States); Siriwardhana, Nalin [Department of Animal Science, The University of Tennessee, Knoxville, TN (United States); Sukhthankar, Mugdha [Department of Biomedical and Diagnostics Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Parra, Columba de la [Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR (United States); Woraratphoka, Jirayus [Department of Obstetrics and Gynecology, Graduate School of Medicine, Medical Center, The University of Tennessee, Knoxville, TN (United States); Malone, Christine [Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Ström, Anders [Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Baek, Seung J. [Department of Biomedical and Diagnostics Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Wade, Paul A. [Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Saxton, Arnold M. [Department of Animal Science, The University of Tennessee, Knoxville, TN (United States); Donnell, Robert M. [Department of Biomedical and Diagnostics Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Pestell, Richard G. [Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA (United States); and others

    2015-02-01

    We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer. - Highlights: • BAD and p-BAD expressions are decreased in breast cancer compared with normal breast tissue. • BAD impedes breast cancer invasion and migration. • BAD inhibits the EMT and transcription factors that promote cancer cell migration. • Invasion and migration functions of BAD are distinct from the BAD's role in apoptosis.

  4. BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion

    International Nuclear Information System (INIS)

    Cekanova, Maria; Fernando, Romaine I.; Siriwardhana, Nalin; Sukhthankar, Mugdha; Parra, Columba de la; Woraratphoka, Jirayus; Malone, Christine; Ström, Anders; Baek, Seung J.; Wade, Paul A.; Saxton, Arnold M.; Donnell, Robert M.; Pestell, Richard G.

    2015-01-01

    We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer. - Highlights: • BAD and p-BAD expressions are decreased in breast cancer compared with normal breast tissue. • BAD impedes breast cancer invasion and migration. • BAD inhibits the EMT and transcription factors that promote cancer cell migration. • Invasion and migration functions of BAD are distinct from the BAD's role in apoptosis

  5. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

    Science.gov (United States)

    Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session.

  6. The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

    Science.gov (United States)

    Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

    2014-02-13

    Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    Science.gov (United States)

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. © 2016 The Protein Society.

  8. Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L.

    Directory of Open Access Journals (Sweden)

    Frédéric Sorgeloos

    Full Text Available Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

  9. Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

    International Nuclear Information System (INIS)

    Jiang, Wen-guo; Zhen, Yong-su; Lu, Xin-an; Shang, Bo-yang; Fu, Yan; Zhang, Sheng-hua; Zhou, Daifu; Li, Liang; Li, Yi; Luo, Yongzhang

    2013-01-01

    Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin

  10. Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

    Science.gov (United States)

    Alam, Parvez; Chaturvedi, Sumit Kumar; Siddiqi, Mohammad Khursheed; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Khan, Rizwan Hasan

    2016-05-27

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

  11. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

    Directory of Open Access Journals (Sweden)

    Leonie Unterholzner

    2011-09-01

    Full Text Available Recognition of viruses by pattern recognition receptors (PRRs causes interferon-β (IFN-β induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1 and IκB kinase-ε (IKKε, which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

  12. Mechanistic Insight into the Host Transcription Inhibition Function of Rift Valley Fever Virus NSs and Its Importance in Virulence.

    Science.gov (United States)

    Terasaki, Kaori; Ramirez, Sydney I; Makino, Shinji

    2016-10-01

    Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the African continent and Middle East. RVFV NSs protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. These include suppression of general transcription, inhibition of IFN-β promoter induction and degradation of double-stranded RNA-dependent protein kinase R. Although each of these biological functions of NSs are considered important for countering the antiviral response in the host, the individual contributions of these functions towards RVFV virulence remains unclear. To examine this, we generated two RVFV MP-12 strain-derived mutant viruses. Each carried mutations in NSs that specifically targeted its general transcription inhibition function without affecting its ability to degrade PKR and inhibit IFN-β promoter induction, through its interaction with Sin3-associated protein 30, a part of the repressor complex at the IFN-β promoter. Using these mutant viruses, we have dissected the transcription inhibition function of NSs and examined its importance in RVFV virulence. Both NSs mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with MP-12. It has been reported that NSs suppresses general transcription by interfering with the formation of the transcription factor IIH complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. Our study results lead us to suggest that the ability of NSs to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. Importantly, RVFV MP-12-NSs mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice

  13. Genetic inhibition of protein kinase Cε attenuates necrosis in experimental pancreatitis

    Science.gov (United States)

    Liu, Yannan; Tan, Tanya; Jia, Wenzhuo; Lugea, Aurelia; Mareninova, Olga; Waldron, Richard T.; Pandol, Stephen J.

    2014-01-01

    Understanding the regulation of death pathways, necrosis and apoptosis, in pancreatitis is important for developing therapies directed to the molecular pathogenesis of the disease. Protein kinase Cε (PKCε) has been previously shown to regulate inflammatory responses and zymogen activation in pancreatitis. Furthermore, we demonstrated that ethanol specifically activated PKCε in pancreatic acinar cells and that PKCε mediated the sensitizing effects of ethanol on inflammatory response in pancreatitis. Here we investigated the role of PKCε in the regulation of death pathways in pancreatitis. We found that genetic deletion of PKCε resulted in decreased necrosis and severity in the in vivo cerulein-induced pancreatitis and that inhibition of PKCε protected the acinar cells from CCK-8 hyperstimulation-induced necrosis and ATP reduction. These findings were associated with upregulation of mitochondrial Bak and Bcl-2/Bcl-xL, proapoptotic and prosurvival members in the Bcl-2 family, respectively, as well as increased mitochondrial cytochrome c release, caspase activation, and apoptosis in pancreatitis in PKCε knockout mice. We further confirmed that cerulein pancreatitis induced a dramatic mitochondrial translocation of PKCε, suggesting that PKCε regulated necrosis in pancreatitis via mechanisms involving mitochondria. Finally, we showed that PKCε deletion downregulated inhibitors of apoptosis proteins, c-IAP2, survivin, and c-FLIPs while promoting cleavage/inactivation of receptor-interacting protein kinase (RIP). Taken together, our findings provide evidence that PKCε activation during pancreatitis promotes necrosis through mechanisms involving mitochondrial proapoptotic and prosurvival Bcl-2 family proteins and upregulation of nonmitochondrial pathways that inhibit caspase activation and RIP cleavage/inactivation. Thus PKCε is a potential target for prevention and/or treatment of acute pancreatitis. PMID:25035113

  14. Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

    Science.gov (United States)

    Xie, Xiaolei; Le, Li; Fan, Yanxin; Lv, Lin; Zhang, Junjie

    2012-07-01

    Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

  15. eIF4A inhibition allows translational regulation of mRNAs encoding proteins involved in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Andrew Bottley

    2010-09-01

    Full Text Available Alzheimer's disease (AD is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta, a cleavage product of amyloid precursor protein (APP. Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.

  16. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-01-01

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe

  17. Protein folding simulations by generalized-ensemble algorithms.

    Science.gov (United States)

    Yoda, Takao; Sugita, Yuji; Okamoto, Yuko

    2014-01-01

    In the protein folding problem, conventional simulations in physical statistical mechanical ensembles, such as the canonical ensemble with fixed temperature, face a great difficulty. This is because there exist a huge number of local-minimum-energy states in the system and the conventional simulations tend to get trapped in these states, giving wrong results. Generalized-ensemble algorithms are based on artificial unphysical ensembles and overcome the above difficulty by performing random walks in potential energy, volume, and other physical quantities or their corresponding conjugate parameters such as temperature, pressure, etc. The advantage of generalized-ensemble simulations lies in the fact that they not only avoid getting trapped in states of energy local minima but also allows the calculations of physical quantities as functions of temperature or other parameters from a single simulation run. In this article we review the generalized-ensemble algorithms. Four examples, multicanonical algorithm, replica-exchange method, replica-exchange multicanonical algorithm, and multicanonical replica-exchange method, are described in detail. Examples of their applications to the protein folding problem are presented.

  18. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  19. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

  20. Injury-induced inhibition of small intestinal protein and nucleic acid synthesis

    International Nuclear Information System (INIS)

    Carter, E.A.; Hatz, R.A.; Yarmush, M.L.; Tompkins, R.G.

    1990-01-01

    Small intestinal mucosal weight and nutrient absorption are significantly diminished early after cutaneous thermal injuries. Because these intestinal properties are highly dependent on rates of nucleic acid and protein synthesis, in vivo incorporation of thymidine, uridine, and leucine into small intestinal deoxyribonucleic acid, ribonucleic acid, and proteins were measured. Deoxyribonucleic acid synthesis was markedly decreased with the lowest thymidine incorporation in the jejunum (p less than 0.01); these findings were confirmed by autoradiographic identification of radiolabeled nuclei in the intestinal crypts. Protein synthesis was decreased by 6 h postinjury (p less than 0.01) but had returned to normal by 48 h. Consistent with a decreased rate of protein synthesis, ribonucleic acid synthesis was also decreased 18 h postinjury (p less than 0.01). These decreased deoxyribonucleic acid, ribonucleic acid, and protein synthesis rates are not likely a result of ischemia because in other studies of this injury model, intestinal blood flow was not significantly changed by the burn injury. Potentially, factors initiating the acute inflammatory reaction may directly inhibit nucleic acid and protein synthesis and lead to alterations in nutrient absorption and intestinal barrier function after injury

  1. Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

    Science.gov (United States)

    Liu, Feng; Roth, Richard A.

    1995-10-01

    To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

  2. Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

    Science.gov (United States)

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  3. Human metapneumovirus M2-2 protein inhibits innate immune response in monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Junping Ren

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS. Whether M2-2 regulates the innate immunity in human dendritic cells (DC, an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2 produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT, suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88, an essential adaptor for Toll-like receptors (TLRs, plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

  4. Mifepristone inhibits extracellular matrix formation in uterine leiomyoma.

    Science.gov (United States)

    Patel, Amrita; Malik, Minnie; Britten, Joy; Cox, Jeris; Catherino, William H

    2016-04-01

    To characterize the efficacy of mifepristone treatment on extracellular matrix (ECM) production in leiomyomas. Laboratory study. University research laboratory. None. Treatment of human immortalized two-dimensional (2D) and three-dimensional (3D) leiomyoma and myometrial cells with mifepristone and the progestin promegestone (R5020). Expression of COL1A1, fibronectin, versican variant V0, and dermatopontin in treated leiomyoma cells by Western blot analysis and confirmatory immunohistochemistry staining of treated 3D cultures. Treatment with progestin stimulated production of COL1A1, fibronectin, versican, and dermatopontin. Mifepristone treatment inhibited protein production of these genes, most notably with versican expression. Combination treatment with both the agonist and antagonist further inhibited protein expression of these genes. Immunohistochemistry performed on 3D cultures demonstrated generalized inhibition of ECM protein concentration. Our study demonstrated that the progesterone agonist R5020 directly stimulated extracellular matrix components COL1A1, fibronectin, versican, and dermatopontin production in human leiomyoma cells. Progesterone antagonist mifepristone decreased protein production of these genes to levels comparable with untreated leiomyoma cells. Published by Elsevier Inc.

  5. Effects on DPPH inhibition of egg-white protein polypeptides treated by pulsed electric field technology.

    Science.gov (United States)

    Wang, Ke; Wang, Jia; Liu, Bolong; Lin, Songyi; Zhao, Ping; Liu, Jingbo; Jones, Gregory; Huang, Hsiang-Chi

    2013-05-01

    Egg-white protein polypeptides are potentially used as a functional ingredient in food products. In this study, the effects on DPPH inhibition of egg-white protein polypeptides ranging from 10 to 30 kDa treated by pulsed electric field (PEF) technology were investigated. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) inhibition (%) was used to evaluate the antioxidant activity of polypeptides. In order to develop and optimize a pulsed electric field (PEF) mathematical model for improving the antioxidant activity, we have investigated three variables, including concentration (6, 8 and 10 mg mL(-1)), electric field intensity (10, 20 and 30 kV cm(-1)) and pulse frequency (2000, 2350 and 2700 Hz) and subsequently optimized them by response surface methodology (RSM). The concentration (8 mg mL(-1)), electric field intensity (10 kV cm(-1)) and pulse frequency (2000 Hz) were found to be the optimal conditions under which the DPPH inhibition increased 28.44%, compared to the sample without PEF treatment. Both near-infrared spectroscopy (NIR) and mid-infrared spectroscopy (MIR) were used to analyze the change of functional groups. The results showed that PEF technology could improve the antioxidant activity of antioxidant polypeptides from egg-white protein under the optimized conditions. © 2012 Society of Chemical Industry.

  6. The antitumor natural product tanshinone IIA inhibits protein kinase C and acts synergistically with 17-AAG.

    Science.gov (United States)

    Lv, Chao; Zeng, Hua-Wu; Wang, Jin-Xin; Yuan, Xing; Zhang, Chuang; Fang, Ting; Yang, Pei-Ming; Wu, Tong; Zhou, Yu-Dong; Nagle, Dale G; Zhang, Wei-Dong

    2018-02-07

    Tanshinone IIA (Tan IIA), the primary bioactive compound derived from the traditional Chinese medicine (TCM) Salvia miltiorrhiza Bunge, has been reported to possess antitumor activity. However, its antitumor mechanisms are not fully understood. To resolve the potential antitumor mechanism(s) of Tan IIA, its gene expression profiles from our database was analyzed by connectivity map (CMAP) and the CMAP-based mechanistic predictions were confirmed/validated in further studies. Specifically, Tan IIA inhibited total protein kinase C (PKC) activity and selectively suppressed the expression of cytosolic and plasma membrane PKC isoforms ζ and ε. The Ras/MAPK pathway that is closely regulated by the PKC signaling is also inhibited by Tan IIA. While Tan IIA did not inhibit heat shock protein 90 (Hsp90), it synergistically enhanced the antitumor efficacy of the Hsp90 inhibitors 17-AAG and ganetespib in human breast cancer MCF-7 cells. In addition, Tan IIA significantly inhibited PI3K/Akt/mTOR signaling, and induced both cell cycle arrest and autophagy. Collectively, these studies provide new insights into the molecular mechanisms responsible for antitumor activity of Tan IIA.

  7. Proteins in the Cocoon of Silkworm Inhibit the Growth of Beauveria bassiana

    Science.gov (United States)

    Zhang, Yan; Li, Youshan; Liu, Huawei; Xia, Qingyou; Zhao, Ping

    2016-01-01

    Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon. PMID:27032085

  8. Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.

    Science.gov (United States)

    Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

    2011-10-01

    Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

  9. Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

    Directory of Open Access Journals (Sweden)

    Tong Zhang

    Full Text Available Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

  10. Simultaneous inhibition of aberrant cancer kinome using rationally designed polymer-protein core-shell nanomedicine.

    Science.gov (United States)

    Chandran, Parwathy; Gupta, Neha; Retnakumari, Archana Payickattu; Malarvizhi, Giridharan Loghanathan; Keechilat, Pavithran; Nair, Shantikumar; Koyakutty, Manzoor

    2013-11-01

    Simultaneous inhibition of deregulated cancer kinome using rationally designed nanomedicine is an advanced therapeutic approach. Herein, we have developed a polymer-protein core-shell nanomedicine to inhibit critically aberrant pro-survival kinases (mTOR, MAPK and STAT5) in primitive (CD34(+)/CD38(-)) Acute Myeloid Leukemia (AML) cells. The nanomedicine consists of poly-lactide-co-glycolide core (~250 nm) loaded with mTOR inhibitor, everolimus, and albumin shell (~25 nm thick) loaded with MAPK/STAT5 inhibitor, sorafenib and the whole construct was surface conjugated with monoclonal antibody against CD33 receptor overexpressed in AML. Electron microscopy confirmed formation of core-shell nanostructure (~290 nm) and flow cytometry and confocal studies showed enhanced cellular uptake of targeted nanomedicine. Simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells, was demonstrated using immunoblotting, cytotoxicity and apoptosis assays. This cell receptor plus multi-kinase targeted core-shell nanomedicine was found better specific and tolerable compared to current clinical regime of cytarabine and daunorubicin. These authors demonstrate simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells by using rationally designed polymer-protein core-shell nanomedicine, provoding an advanced method to eliminate cancer cells, with the hope of future therapeutic use. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. The bisphosphonate zoledronic acid effectively targets lung cancer cells by inhibition of protein prenylation

    International Nuclear Information System (INIS)

    Xie, Fan; Li, Pengcheng; Gong, Jianhua; Zhang, Jiahong; Ma, Jingping

    2015-01-01

    Aberrant activation of oncoproteins such as members of the Ras family is common in human lung cancers. The proper function of Ras largely depends on a post-translational modification termed prenylation. Bisphosphonates have been shown to inhibit prenylation in cancer cells. In this study, we show that zoledronic acid, a third generation bisphosphonate, is effective in targeting lung cancer cells. This is achieved by the induction of apoptosis and inhibition of proliferation, through suppressing the activation of downstream Ras and EGFR signalling by zoledronic acid. The combination of zoledronic acid and paclitaxel or cisplatin (commonly used chemotherapeutic drugs for lung cancer) augmented the activity of either drug alone in in vitro lung cancer cellular system and in vivo lung xenograft mouse model. Importantly, zoledronic acid inhibits protein prenylation as shown by the increased levels of unprenylated Ras and Rap1A. In addition, the effects of zoledronic acid were reversed in the presence of geranylgeraniol and farnesol, further confirming that mechanism of zoledroinc acid's action in lung cancer cells is through prenylation inhibition. Since zoledronic acid is already available for clinic use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of lung cancer. - Highlights: • Zoledronic acid (ZA) is effectively against lung cancer cells in vitro and in vivo. • ZA acts on lung cancer cells through inhibition of protein prenylation. • ZA suppresses global downstream phosphorylation of Ras signalling. • ZA enhances the effects of chemotherapeutic drugs in lung cancer cells.

  12. The bisphosphonate zoledronic acid effectively targets lung cancer cells by inhibition of protein prenylation

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Fan [Department of Respiratory Medicine, Jingzhou Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Jingzhou (China); Li, Pengcheng [Department of Oncology, Wuhan Union Hospital Affiliated to Huazhong University of Science and Technology, Wuhan (China); Gong, Jianhua; Zhang, Jiahong [Department of Respiratory Medicine, Jingzhou Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Jingzhou (China); Ma, Jingping, E-mail: mjpjzhospital@hotmail.com [Department of Respiratory Medicine, Jingzhou Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Jingzhou (China)

    2015-11-27

    Aberrant activation of oncoproteins such as members of the Ras family is common in human lung cancers. The proper function of Ras largely depends on a post-translational modification termed prenylation. Bisphosphonates have been shown to inhibit prenylation in cancer cells. In this study, we show that zoledronic acid, a third generation bisphosphonate, is effective in targeting lung cancer cells. This is achieved by the induction of apoptosis and inhibition of proliferation, through suppressing the activation of downstream Ras and EGFR signalling by zoledronic acid. The combination of zoledronic acid and paclitaxel or cisplatin (commonly used chemotherapeutic drugs for lung cancer) augmented the activity of either drug alone in in vitro lung cancer cellular system and in vivo lung xenograft mouse model. Importantly, zoledronic acid inhibits protein prenylation as shown by the increased levels of unprenylated Ras and Rap1A. In addition, the effects of zoledronic acid were reversed in the presence of geranylgeraniol and farnesol, further confirming that mechanism of zoledroinc acid's action in lung cancer cells is through prenylation inhibition. Since zoledronic acid is already available for clinic use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of lung cancer. - Highlights: • Zoledronic acid (ZA) is effectively against lung cancer cells in vitro and in vivo. • ZA acts on lung cancer cells through inhibition of protein prenylation. • ZA suppresses global downstream phosphorylation of Ras signalling. • ZA enhances the effects of chemotherapeutic drugs in lung cancer cells.

  13. Protein Polymers and Amyloids

    DEFF Research Database (Denmark)

    Risør, Michael Wulff

    2014-01-01

    Several human disorders are caused by a common general disease mechanism arising from abnormal folding and aggregation of the underlying protein. These include the prevalent dementias like Alzheimer’s and Parkinson’s, where accumulation of protein fibrillar structures, known as amyloid fibrils......, is a general hallmark. They also include the α1-antitrypsin deficiency, where disease-causing mutations in the serine protease inhibitor, α1-antitrypsin (α1AT), leads to accumulation of the aberrant protein in the liver of these patients. The native metastable structure of α1AT constitutes a molecular trap...... that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecular level is not only important for our general knowledge on protein folding...

  14. Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

    International Nuclear Information System (INIS)

    Saintigny, Yannick

    1999-01-01

    The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author) [fr

  15. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis...

  16. Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

    International Nuclear Information System (INIS)

    Warters, R.L.; Brizgys, L.M.; Lyons, B.W.

    1987-01-01

    The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 0 C and 45 0 C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 0 C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 37 0 C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 0 C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

  17. Inhibition of H9N2 virus invasion into dendritic cells by the S-layer protein from L. acidophilus ATCC 4356

    Directory of Open Access Journals (Sweden)

    Xue Gao

    2016-10-01

    Full Text Available Probiotics are essential for the prevention of virus invasion and the maintenance of the immune balance. However, the mechanism of competition between probiotics and virus are unknown. The objectives of this study were to isolate the surface layer (S-layer protein from L. acidophilus ATCC 4356 as a new antiviral material, to evaluate the stimulatory effects of the S-layer protein on mouse dendritic cells (DCs and to verify its ability to inhibit the invasion of H9N2 avian influenza virus (AIV in DCs. We found that the S-layer protein induced DCs activation and up-regulated the IL-10 secretion. The invasion and replication of the H9N2 virus in mouse DCs was successfully demonstrated. However, the invasion of H9N2 virus into DCs could be inhibited by treatment with the S-layer protein prior to infection, which was verified by the reduced hemagglutinin (HA and neuraminidase (NA mRNA expression, and nucleoprotein (NP protein expression in the DCs. Furthermore, treatment with the S-layer protein increases the Mx1, Isg15, and Ddx58 mRNA expressions, and remits the inflammatory process to inhibit H9N2 AIV infection. In conclusion, the S-layer protein stimulates the activation of mouse DCs, inhibits H9N2 virus invasion of DCs, and stimulates the IFN-I signalling pathway. Thus, the S-layer protein from Lactobacillus is a promising biological antiviral material for AIV prevention.

  18. Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

    Science.gov (United States)

    Kuschal, Christiane; Thoms, Kai-Martin; Boeckmann, Lars; Laspe, Petra; Apel, Antje; Schön, Michael P; Emmert, Steffen

    2011-10-01

    Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner. © 2011 John Wiley & Sons A/S.

  19. Hantaan Virus Nucleocapsid Protein Binds to Importin alpha Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B

    Science.gov (United States)

    2008-11-19

    Microbiology . All Rights Reserved. Hantaan Virus Nucleocapsid Protein Binds to Importin Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced...Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702,1 and Department of Microbiology , Mount Sinai...34–36. 32. Prescott , J., C. Ye, G. Sen, and B. Hjelle. 2005. Induction of innate immune response genes by Sin Nombre hantavirus does not require

  20. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    International Nuclear Information System (INIS)

    Honma, Yuichi; Harada, Masaru

    2013-01-01

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation

  1. Inhibition of HCV replication by oxysterol-binding protein-related protein 4 (ORP4 through interaction with HCV NS5B and alteration of lipid droplet formation.

    Directory of Open Access Journals (Sweden)

    In-Woo Park

    Full Text Available Hepatitis C virus (HCV RNA replication involves complex interactions among the 3'x RNA element within the HCV 3' untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3' X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4, a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.

  2. Lactococcal Abortive Infection Protein AbiV Interacts Directly with the Phage Protein SaV and Prevents Translation of Phage Proteins

    DEFF Research Database (Denmark)

    Haaber, Jakob Brandt Borup; Samson, J.E.; Labrie, S.J.

    2010-01-01

    RNAs and proteins suggested that AbiV blocks the activation of late gene transcription, probably by a general inhibition of translation. Using size exclusion chromatography coupled with on-line static light scattering and refractometry, as well as fluorescence quenching experiments, we also demonstrated that both...

  3. Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Long, Cong; Wang, Jingchao [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Guo, Wei [Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Wang, Huan; Wang, Chao; Liu, Yu [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Sun, Xiaoping, E-mail: xsun6@whu.edu.cn [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); State Key Laboratory of Virology, Wuhan University, Wuhan, 430072 (China)

    2016-01-01

    Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated that triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.

  4. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    International Nuclear Information System (INIS)

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    2016-01-01

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B"c"a expression did not restore adipogenesis.

  5. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Kadri, Zahra; Granger-Locatelli, Marine [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Fucharoen, Suthat [Thalassemia Research Center, Mahidol University (Thailand); Maouche-Chrétien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France); Prost, Stéphane [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Chrétien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France)

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B{sup ca} expression did not restore adipogenesis.

  6. Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

    Science.gov (United States)

    Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

    2012-10-26

    The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

  7. Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway

    Science.gov (United States)

    Li, Dan; Lei, Caoqi; Xu, Zhisheng; Yang, Fan; Liu, Huanan; Zhu, Zixiang; Li, Shu; Liu, Xiangtao; Shu, Hongbing; Zheng, Haixue

    2016-01-01

    Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects cloven-hoofed animals. The pathophysiology of FMDV has not been fully understood and the evasion of host innate immune system is still unclear. Here, the FMDV non-structural protein 3A was identified as a negative regulator of virus-triggered IFN-β signaling pathway. Overexpression of the FMDV 3A inhibited Sendai virus-triggered activation of IRF3 and the expressions of RIG-I/MDA5. Transient transfection and co-immunoprecipitation experiments suggested that FMDV 3A interacts with RIG-I, MDA5 and VISA, which is dependent on the N-terminal 51 amino acids of 3A. Furthermore, 3A also inhibited the expressions of RIG-I, MDA5, and VISA by disrupting their mRNA levels. These results demonstrated that 3A inhibits the RLR-mediated IFN-β induction and uncovered a novel mechanism by which the FMDV 3A protein evades the host innate immune system. PMID:26883855

  8. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Scaife, R.M. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Wilson, L. (Univ. of California, Santa Barbara (United States)); Purich, D.L. (Univ. of Florida, Gainesville (United States))

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  9. Digestibility of Quinoa (Chenopodium quinoa Willd.) Protein Concentrate and Its Potential to Inhibit Lipid Peroxidation in the Zebrafish Larvae Model.

    Science.gov (United States)

    Vilcacundo, R; Barrio, D; Carpio, C; García-Ruiz, A; Rúales, J; Hernández-Ledesma, B; Carrillo, W

    2017-09-01

    Quinoa protein concentrate (QPC) was extracted and digested under in vitro gastrointestinal conditions. The protein content of QPC was in the range between 52.40 and 65.01% depending on the assay used. Quinoa proteins were almost completely hydrolyzed by pepsin at pH of 1.2, 2.0, and 3.2. At high pH, only partial hydrolysis was observed. During the duodenal phase, no intact proteins were visible, indicating their susceptibility to the in vitro simulated digestive conditions. Zebrafish larvae model was used to evaluate the in vivo ability of gastrointestinal digests to inhibit lipid peroxidation. Gastric digestion at pH 1.2 showed the highest lipid peroxidation inhibition percentage (75.15%). The lipid peroxidation activity increased after the duodenal phase. The digest obtained at the end of the digestive process showed an inhibition percentage of 82.10%, comparable to that showed when using BHT as positive control (87.13%).

  10. The dynamical mechanism of auto-inhibition of AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Cheng Peng

    2011-07-01

    Full Text Available We use a novel normal mode analysis of an elastic network model drawn from configurations generated during microsecond all-atom molecular dynamics simulations to analyze the mechanism of auto-inhibition of AMP-activated protein kinase (AMPK. A recent X-ray and mutagenesis experiment (Chen, et al Nature 2009, 459, 1146 of the AMPK homolog S. Pombe sucrose non-fermenting 1 (SNF1 has proposed a new conformational switch model involving the movement of the kinase domain (KD between an inactive unphosphorylated open state and an active or semi-active phosphorylated closed state, mediated by the autoinhibitory domain (AID, and a similar mutagenesis study showed that rat AMPK has the same auto-inhibition mechanism. However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited. By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition. Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop.

  11. Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11.

    Science.gov (United States)

    Li, Manqing; Kao, Elaine; Gao, Xia; Sandig, Hilary; Limmer, Kirsten; Pavon-Eternod, Mariana; Jones, Thomas E; Landry, Sebastien; Pan, Tao; Weitzman, Matthew D; David, Michael

    2012-11-01

    In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination.

  12. Model for Stress-induced Protein Degradation in Lemna minor1

    Science.gov (United States)

    Cooke, Robert J.; Roberts, Keith; Davies, David D.

    1980-01-01

    Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

  13. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Science.gov (United States)

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

  14. Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

    Science.gov (United States)

    Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

    2011-06-03

    Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

  15. Dynamic features of apo and bound HIV-Nef protein reveal the anti-HIV dimerization inhibition mechanism.

    Science.gov (United States)

    Moonsamy, Suri; Bhakat, Soumendranath; Soliman, Mahmoud E S

    2015-01-01

    The first account on the dynamic features of Nef or negative factor, a small myristoylated protein located in the cytoplasm believes to increase HIV-1 viral titer level, is reported herein. Due to its major role in HIV-1 pathogenicity, Nef protein is considered an emerging target in anti-HIV drug design and discovery process. In this study, comparative long-range all-atom molecular dynamics simulations were employed for apo and bound protein to unveil molecular mechanism of HIV-Nef dimerization and inhibition. Results clearly revealed that B9, a newly discovered Nef inhibitor, binds at the dimeric interface of Nef protein and caused significant separation between orthogonally opposed residues, namely Asp108, Leu112 and Gln104. Large differences in magnitudes were observed in the radius of gyration (∼1.5 Å), per-residue fluctuation (∼2 Å), C-alpha deviations (∼2 Å) which confirm a comparatively more flexible nature of apo conformation due to rapid dimeric association. Compared to the bound conformer, a more globally correlated motion in case of apo structure of HIV-Nef confirms the process of dimeric association. This clearly highlights the process of inhibition as a result of ligand binding. The difference in principal component analysis (PCA) scatter plot and per-residue mobility plot across first two normal modes further justifies the same findings. The in-depth dynamic analyses of Nef protein presented in this report would serve crucial in understanding its function and inhibition mechanisms. Information on inhibitor binding mode would also assist in designing of potential inhibitors against this important HIV target.

  16. Toward Small-Molecule Inhibition of Protein-Protein Interactions: General Aspects and Recent Progress in Targeting Costimulatory and Coinhibitory (Immune Checkpoint) Interactions.

    Science.gov (United States)

    Bojadzic, Damir; Buchwald, Peter

    2018-05-30

    Protein-protein interactions (PPIs) that are part of the costimulatory and coinhibitory (immune checkpoint) signaling are critical for adequate T cell response and are important therapeutic targets for immunomodulation. Biologics targeting them have already achieved considerable clinical success in the treatment of autoimmune diseases or transplant recipients (e.g., abatacept, belatacept, and belimumab) as well as cancer (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab). In view of such progress, there have been only relatively limited efforts toward developing small-molecule PPI inhibitors (SMPPIIs) targeting these cosignaling interactions, possibly because they, as all other PPIs, are difficult to target by small molecules and were not considered druggable. Nevertheless, substantial progress has been achieved during the last decade. SMPPIIs proving the feasibility of such approaches have been identified through various strategies for a number of cosignaling interactions including CD40-CD40L, OX40-OX40L, BAFFR-BAFF, CD80-CD28, and PD-1-PD-L1s. Here, after an overview of the general aspects and challenges of SMPPII-focused drug discovery, we review them briefly together with relevant structural, immune-signaling, physicochemical, and medicinal chemistry aspects. While so far only a few of these SMPPIIs have shown activity in animal models (DRI-C21045 for CD40-D40L, KR33426 for BAFFR-BAFF) or reached clinical development (RhuDex for CD80-CD28, CA-170 for PD-1-PD-L1), there is proof-of-principle evidence for the feasibility of such approaches in immunomodulation. They can result in products that are easier to develop/manufacture and are less likely to be immunogenic or encounter postmarket safety events than corresponding biologics, and, contrary to them, can even become orally bioavailable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    Science.gov (United States)

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

    International Nuclear Information System (INIS)

    Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

    2011-01-01

    Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+ ); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+ . Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+ -induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

  19. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    Science.gov (United States)

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  20. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Ciomperlik, Jessica J. [Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 (United States); Basta, Holly A. [Department of Biology, Rocky Mountain College, Billings, MT (United States); Palmenberg, Ann C., E-mail: acpalmen@wisc.edu [Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 (United States)

    2016-01-15

    Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced to alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.

  1. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    Science.gov (United States)

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. On the characterization and software implementation of general protein lattice models.

    Directory of Open Access Journals (Sweden)

    Alessio Bechini

    Full Text Available models of proteins have been widely used as a practical means to computationally investigate general properties of the system. In lattice models any sterically feasible conformation is represented as a self-avoiding walk on a lattice, and residue types are limited in number. So far, only two- or three-dimensional lattices have been used. The inspection of the neighborhood of alpha carbons in the core of real proteins reveals that also lattices with higher coordination numbers, possibly in higher dimensional spaces, can be adopted. In this paper, a new general parametric lattice model for simplified protein conformations is proposed and investigated. It is shown how the supporting software can be consistently designed to let algorithms that operate on protein structures be implemented in a lattice-agnostic way. The necessary theoretical foundations are developed and organically presented, pinpointing the role of the concept of main directions in lattice-agnostic model handling. Subsequently, the model features across dimensions and lattice types are explored in tests performed on benchmark protein sequences, using a Python implementation. Simulations give insights on the use of square and triangular lattices in a range of dimensions. The trend of potential minimum for sequences of different lengths, varying the lattice dimension, is uncovered. Moreover, an extensive quantitative characterization of the usage of the so-called "move types" is reported for the first time. The proposed general framework for the development of lattice models is simple yet complete, and an object-oriented architecture can be proficiently employed for the supporting software, by designing ad-hoc classes. The proposed framework represents a new general viewpoint that potentially subsumes a number of solutions previously studied. The adoption of the described model pushes to look at protein structure issues from a more general and essential perspective, making

  3. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    Energy Technology Data Exchange (ETDEWEB)

    McCready, Jessica [Department of Natural Sciences, Assumption College, Worcester, MA 01609 (United States); Wong, Daniel S. [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States); Burlison, Joseph A.; Ying, Weiwen [Synta Pharmaceuticals, Lexington, MA 02421 (United States); Jay, Daniel G., E-mail: daniel.jay@tufts.edu [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States)

    2014-04-30

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

  4. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    International Nuclear Information System (INIS)

    McCready, Jessica; Wong, Daniel S.; Burlison, Joseph A.; Ying, Weiwen; Jay, Daniel G.

    2014-01-01

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion

  5. Arctigenin inhibits triple-negative breast cancers by targeting CIP2A to reactivate protein phosphatase 2A.

    Science.gov (United States)

    Huang, Qiuyue; Qin, Shanshan; Yuan, Xiaoning; Zhang, Liang; Ji, Juanli; Liu, Xuewen; Ma, Wenjing; Zhang, Yunfei; Liu, Pengfei; Sun, Zhiting; Zhang, Jingxuan; Liu, Ying

    2017-07-01

    We have shown that a novel STAT3 inhibitor arctigenin (Atn) induces significant cytotoxicity in triple-negative breast cancer (TNBC) cells. This study further delineated molecular mechanisms where by Atn triggered cytotoxicity in TNBC cells. We found Atn can also inhibit metastasis in TNBC cells through cancerous inhibitor of protein phosphatase 2A (CIP2A) pathway. CIP2A is an endogenous inhibitor of protein phosphatase 2A (PP2A), which can increase the migration and invasion of various cancer cells. PP2A is a tumor suppressor, which is functionally defective in various cancers. Atn-induced metastasis inhibition was associated with reactivation of PP2A, downregulation of CIP2A and Akt phosphorylation. Silencing CIP2A enhanced Atn-induced metastasis inhibition and apoptosis in TNBCs. Furthermore, ectopic expression of CIP2A or inhibition of PP2A in TNBC cells abolished the effects of Atn. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A, at least in part, promotes the anti-metastasis effect induced by Atn. Our findings disclose the novel therapeutic mechanism of this targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.

  6. Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

    Science.gov (United States)

    Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

    2017-11-15

    A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in

  7. Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

    Science.gov (United States)

    Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

    2004-10-01

    We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

  8. Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki; Rhee, David Y.; Connelly, Michele; Sviderskiy, Vladislav O.; Bhasin, Deepak; Chen, Yizhe; Ong, Su-Sien; Chai, Sergio C.; Goktug, Asli N.; Huang, Guochang; Monda, Julie K.; Low, Jonathan; Kim, Ho Shin; Paulo, Joao A.; Cannon, Joe R.; Shelat, Anang A.; Chen, Taosheng; Kelsall, Ian R.; Alpi, Arno F.; Pagala, Vishwajeeth; Wang, Xusheng; Peng, Junmin; Singh , Bhuvanesh; Harper, J. Wade; Schulman, Brenda A.; Guy, R. Kip (MSKCC); (Dundee); (SJCH); (Harvard-Med); (MXPL)

    2017-06-05

    N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.

  9. Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

    Science.gov (United States)

    Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori

    2012-02-01

    Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function

  10. Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells

    International Nuclear Information System (INIS)

    Sato, Naoki; Maeda, Masao; Sugiyama, Mai; Ito, Satoko; Hyodo, Toshinori; Masuda, Akio; Tsunoda, Nobuyuki; Kokuryo, Toshio; Hamaguchi, Michinari; Nagino, Masato; Senga, Takeshi

    2015-01-01

    RNA splicing is a fundamental process for protein synthesis. Recent studies have reported that drugs that inhibit splicing have cytotoxic effects on various tumor cell lines. In this report, we demonstrate that depletion of SNW1, a component of the spliceosome, induces apoptosis in breast cancer cells. Proteomics and biochemical analyses revealed that SNW1 directly associates with other spliceosome components, including EFTUD2 (Snu114) and SNRNP200 (Brr2). The SKIP region of SNW1 interacted with the N-terminus of EFTUD2 as well as two independent regions in the C-terminus of SNRNP200. Similar to SNW1 depletion, knockdown of EFTUD2 increased the numbers of apoptotic cells. Furthermore, we demonstrate that exogenous expression of either the SKIP region of SNW1 or the N-terminus region of EFTUD2 significantly promoted cellular apoptosis. Our results suggest that the inhibition of SNW1 or its associating proteins may be a novel therapeutic strategy for cancer treatment

  11. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    International Nuclear Information System (INIS)

    Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na; Guo, Qinglong

    2013-01-01

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  12. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na, E-mail: luna555@163.com; Guo, Qinglong, E-mail: anticancer_drug@yahoo.com.cn

    2013-09-01

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  13. In silico analysis of the polygalacturonase inhibiting protein 1 from apple, Malus domestica.

    Science.gov (United States)

    Matsaunyane, Lerato Bt; Oelofse, Dean; Dubery, Ian A

    2015-03-11

    The Malus domestica polygalacturonase inhibiting protein 1 (MdPGIP1) gene, encoding the M. domestica polygalacturonase inhibiting protein 1 (MdPGIP1), was isolated from the Granny Smith apple cultivar (GenBank accession no. DQ185063). The gene was used to transform tobacco and potato for enhanced resistance against fungal diseases. Analysis of the MdPGIP1 nucleotide sequence revealed that the gene comprises 993 nucleotides that encode a 330 amino acid polypeptide. In silico characterization of the MdPGIP1 polypeptide revealed domains typical of PGIP proteins, which include a 24 amino acid putative signal peptide, a potential cleavage site [Alanine-Leucine-Serine (ALS)] for the signal peptide, a 238 amino acid leucine-rich repeat (LRR) domain, a 46 amino acid N-terminal domain and a 22 amino acid C-terminal domain. The hydropathic evaluation of MdPGIP1 indicated a repetitive hydrophobic motif in the LRR domain and a hydrophilic surface area consistent with a globular protein. The typical consensus glycosylation sequence of Asn-X-Ser/Thr was identified in MdPGIP1, indicating potential N-linked glycosylation of MdPGIP1. The molecular mass of non-glycosylated MdPGIP1 was calculated as 36.615 kDa and the theoretical isoelectric point as 6.98. Furthermore, the secondary and tertiary structure of MdPGIP1 was modelled, and revealed that MdPGIP1 is a curved and elongated molecule that contains sheet B1, sheet B2 and 310-helices on its LRR domain. The overall properties of the MdPGIP1 protein is similar to that of the prototypical Phaseolus vulgaris PGIP 2 (PvPGIP2), and the detected differences supported its use in biotechnological applications as an inhibitor of targeted fungal polygalacturonases (PGs).

  14. Inhibition of interleukin-6 expression by the V protein of parainfluenza virus 5

    International Nuclear Information System (INIS)

    Lin Yuan; Sun Minghao; Fuentes, Sandra M.; Keim, Celia D.; Rothermel, Terri; He Biao

    2007-01-01

    The V protein of parainfluenza virus 5 (PIV5) plays an important role in the evasion of host immune responses. The V protein blocks interferon (IFN) signaling in human cells by causing degradation of the STAT1 protein, a key component of IFN signaling, and blocks IFN-β production by preventing nuclear translocation of IRF3, a key transcription factor for activating IFN-β promoter. Interleukin-6 (IL-6), along with tumor necrosis factor (TNF)-α and IL-1β, is a major proinflammatory cytokine that plays important roles in clearing virus infection through inflammatory responses. Many viruses have developed strategies to block IL-6 expression. Wild-type PIV5 infection induces little, if any, expression of cytokines such as IL-6 or TNF-α, whereas infection by a mutant PIV5 lacking the conserved C-terminal cysteine rich domain (rPIV5VΔC) induced high levels of IL-6 expression. Examination of mRNA levels of IL-6 indicated that the transcription activation of IL-6 played an important role in the increased IL-6 expression. Co-infection with wild-type PIV5 prevented the activation of IL-6 transcription by rPIV5VΔC, and a plasmid encoding the full-length PIV5 V protein prevented the activation of IL-6 promoter-driven reporter gene expression by rPIV5VΔC, indicating that the V protein played a role in inhibiting IL-6 transcription. The activation of IL-6 was independent of IFN-β even though rPIV5VΔC-infected cells produced IFN-β. Using reporter gene assays and chromatin immunoprecipitation (ChIP), it was found that NF-κB played an important role in activating expression of IL-6. We have proposed a model of activating and inhibiting IL-6 transcription by PIV5

  15. Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma.

    Science.gov (United States)

    Markowitz, Daniel; Powell, Caitlin; Tran, Nhan L; Berens, Michael E; Ryken, Timothy C; Vanan, Magimairajan; Rosen, Lisa; He, Mingzu; Sun, Shan; Symons, Marc; Al-Abed, Yousef; Ruggieri, Rosamaria

    2016-08-01

    Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiotherapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell-cycle arrest after ionizing radiation. Here, we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a specific small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radiosensitizes UW228 medulloblastoma cells as well as UI226 patient-derived primary cells, whereas it does not affect the response to radiation of normal brain cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell-cycle arrest. Importantly, in an animal model of medulloblastoma that employs orthotopic implantation of primary patient-derived UI226 cells in nude mice, M443 in combination with radiation achieved a synergistic increase in survival. We hypothesize that combining radiotherapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Mol Cancer Ther; 15(8); 1799-808. ©2016 AACR. ©2016 American Association for Cancer Research.

  16. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    Energy Technology Data Exchange (ETDEWEB)

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  17. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    International Nuclear Information System (INIS)

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-01-01

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR γ ) and CCAAT element binding protein α (C/EBP α ), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins

  18. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARγ Expression and Activation in Differentiating Mesenchymal Stem Cells

    Science.gov (United States)

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARγ2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARγ, and SREBP-1 were determined by western blot. Finally, DNA binding PPARγ activity was determined using an ELISA-based PPARγ activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARγ expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARγ activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARγ expression and activity. PMID:18274630

  19. Chloroquine inhibits accessory cell presentation of soluble natural and synthetic protein antigens

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...... was time- and dose-dependent. A brief treatment solely of the accessory cells with the drug compromised their ability to stimulate primed T cells in a subsequent culture provided the accessory cells were treated with chloroquine before their exposure to the antigen. These results suggest that chloroquine...... acts on an early event in the antigen handling by accessory cells. Chloroquine is a well known inhibitor of lysosomal proteolysis, and it is likely that its effect on antigen presentation is caused by an inhibition of antigen degradation....

  20. NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery.

    Science.gov (United States)

    Barry, Gerald; Varela, Mariana; Ratinier, Maxime; Blomström, Anne-Lie; Caporale, Marco; Seehusen, Frauke; Hahn, Kerstin; Schnettler, Esther; Baumgärtner, Wolfgang; Kohl, Alain; Palmarini, Massimo

    2014-08-01

    Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways. © 2014 The Authors.

  1. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication

    International Nuclear Information System (INIS)

    Xiong Wei; Wang Xun; Liu Xiaoying; Xiang Li; Zheng Lingjie; Yuan Zhenghong

    2004-01-01

    Myeloid differential primary response protein (MyD88) is a critical component in the signaling cascade through Toll-like receptors (TLRs) and is induced by α interferon (IFN-α). To examine the role of MyD88 in the antiviral activity of IFN-α against hepatitis B virus (HBV), we established MyD88 stably expressing cell lines and studied HBV replication in these lines after transient transfection. The levels of HBV proteins and viral replicative intermediates were effectively reduced in MyD88-expressing cells. A significant reduction of total and cytoplasmic viral RNAs in MyD88 stably expressing cells was also observed. Using a nuclear factor-κB (NF-κB) dependent reporter assay, it was shown that activation of NF-κB was moderately increased in the presence of expression of MyD88, and further significantly increased by co-expression of HBV. These results suggest a novel mechanism for the inhibition of HBV replication by IFN-α via expression of MyD88 protein involving activation of NF-κB signaling pathway and downregulation of viral transcription

  2. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    International Nuclear Information System (INIS)

    Xi, Dong; Dong, Xiao; Deng, Wei; Lai, Luhua

    2011-01-01

    Highlights: ► Mechanism of small heat shock protein inhibition on fibril formation was studied. ► Peptide SSTSAA with modified ends was used for amyloid fibril formation. ► FRET signal was followed during the fibril formation. ► Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. ► Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  3. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Dong [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Center for Theoretical Biology, Peking University, Beijing 100871 (China); Dong, Xiao; Deng, Wei [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Lai, Luhua, E-mail: lhlai@pku.edu.cn [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Center for Theoretical Biology, Peking University, Beijing 100871 (China)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mechanism of small heat shock protein inhibition on fibril formation was studied. Black-Right-Pointing-Pointer Peptide SSTSAA with modified ends was used for amyloid fibril formation. Black-Right-Pointing-Pointer FRET signal was followed during the fibril formation. Black-Right-Pointing-Pointer Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. Black-Right-Pointing-Pointer Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  4. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-02-01

    Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

  5. FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit.

    Science.gov (United States)

    Bibert, Stéphanie; Liu, Chia-Chi; Figtree, Gemma A; Garcia, Alvaro; Hamilton, Elisha J; Marassi, Francesca M; Sweadner, Kathleen J; Cornelius, Flemming; Geering, Käthi; Rasmussen, Helge H

    2011-05-27

    The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

  6. The structure of a contact-dependent growth-inhibition (CDI) immunity protein from Neisseria meningitidis MC58

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Kemin; Johnson, Parker M.; Stols, Lucy; Boubion, Bryan; Eschenfeldt, William; Babnigg, Gyorgy; Hayes, Christopher S.; Joachimiak, Andrezj; Goulding, Celia W.

    2015-05-20

    Contact-dependent growth inhibition (CDI) is an important mechanism of intercellular competition between neighboring Gram-negative bacteria. CDI systems encode large surface-exposed CdiA effector proteins that carry a variety of C-terminal toxin domains (CdiA-CTs). All CDI+bacteria also produce CdiI immunity proteins that specifically bind to the cognate CdiA-CT and neutralize its toxin activity to prevent auto-inhibition. Here, the X-ray crystal structure of a CdiI immunity protein fromNeisseria meningitidisMC58 is presented at 1.45 Å resolution. The CdiI protein has structural homology to the Whirly family of RNA-binding proteins, but appears to lack the characteristic nucleic acid-binding motif of this family. Sequence homology suggests that the cognate CdiA-CT is related to the eukaryotic EndoU family of RNA-processing enzymes. A homology model is presented of the CdiA-CT based on the structure of the XendoU nuclease fromXenopus laevis. Molecular-docking simulations predict that the CdiA-CT toxin active site is occluded upon binding to the CdiI immunity protein. Together, these observations suggest that the immunity protein neutralizes toxin activity by preventing access to RNA substrates.

  7. Asynchronous Cholinergic Drive Correlates with Excitation-Inhibition Imbalance via a Neuronal Ca2+ Sensor Protein

    Directory of Open Access Journals (Sweden)

    Keming Zhou

    2017-05-01

    Full Text Available Excitation-inhibition imbalance in neural networks is widely linked to neurological and neuropsychiatric disorders. However, how genetic factors alter neuronal activity, leading to excitation-inhibition imbalance, remains unclear. Here, using the C. elegans locomotor circuit, we examine how altering neuronal activity for varying time periods affects synaptic release pattern and animal behavior. We show that while short-duration activation of excitatory cholinergic neurons elicits a reversible enhancement of presynaptic strength, persistent activation results to asynchronous and reduced cholinergic drive, inducing imbalance between endogenous excitation and inhibition. We find that the neuronal calcium sensor protein NCS-2 is required for asynchronous cholinergic release in an activity-dependent manner and dampens excitability of inhibitory neurons non-cell autonomously. The function of NCS-2 requires its Ca2+ binding and membrane association domains. These results reveal a synaptic mechanism implicating asynchronous release in regulation of excitation-inhibition balance.

  8. YeeV is an Escherichia coli toxin that inhibits cell division by targeting the cytoskeleton proteins, FtsZ and MreB.

    Science.gov (United States)

    Tan, Qian; Awano, Naoki; Inouye, Masayori

    2011-01-01

    Toxin-antitoxin (TA) systems of free-living bacteria have recently demonstrated that these toxins inhibit cell growth by targeting essential functions of cellular metabolism. Here we show that YeeV toxin inhibits cell division, leads to a change in morphology and lysis of Escherichia coli cells. YeeV interacts with two essential cytoskeleton proteins, FtsZ and MreB. Purified YeeV inhibits both the GTPase activity and the GTP-dependent polymerization of FtsZ. YeeV also inhibits ATP-dependent polymerization of MreB. Truncated C-terminal deletions of YeeV result in elongation of cells, and a deletion of the first 15 amino acids from the N-terminus of YeeV caused lemon-shaped cell formation. The YeeV toxin is distinct from other well-studied toxins: it directs the binding of two cytoskeletal proteins and inhibits FtsZ and MreB simultaneously. © 2010 Blackwell Publishing Ltd.

  9. The murine gammaherpesvirus-68 chemokine-binding protein M3 inhibits experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Millward, Jason M; Holst, Peter J; Høgh-Petersen, Mette

    2010-01-01

    M3 (AdM3) directly to the CNS to evaluate the capacity of this protein to inhibit neuroinflammation using the experimental autoimmune encephalomyelitis (EAE) model. Treatment with the AdM3 vector significantly reduced the clinical severity of EAE, attenuated CNS histopathology, and reduced numbers......Chemokines are critical mediators of immune cell entry into the central nervous system (CNS), as occurs in neuroinflammatory disease such as multiple sclerosis. Chemokines are also implicated in the immune response to viral infections. Many viruses encode proteins that mimic or block chemokine...... of immune cells infiltrating the CNS. These results suggest that M3 may represent a novel therapeutic approach to neuroinflammatory disease....

  10. Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Nana Liu

    Full Text Available Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species. Polygalacturonase-inhibiting proteins (PGIPs are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7-66.4% and 56.5-60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.

  11. Properties of a polygalacturonase-inhibiting protein isolated from 'Oroblanco' grapefruit.

    Science.gov (United States)

    D'hallewin, Guy; Schirra, Mario; Powell, Ann L. T.; Greve, L. Carl; Labavitch, John M.

    2004-03-01

    Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4 degrees C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 mM alpha-methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea. The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum, growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65 degrees C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100 degrees C for 10 min.

  12. Extracellular fibrinogen-binding protein (Efb) from staphylococcus aureus Inhibits the formation of platelet-leukocyte complexes

    NARCIS (Netherlands)

    Posner, M.G; Upadhyay, A.; Abubaker, A.A.; Fortunato, T.M.; Vara, D.; Canobbio, I.; Bagby, S.; Pula, G.

    2016-01-01

    Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to

  13. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate......-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate...

  14. Assessment of Novel Anti-thrombotic Fusion Proteins for Inhibition of Stenosis in a Porcine Model of Arteriovenous Graft.

    Directory of Open Access Journals (Sweden)

    Christi M Terry

    Full Text Available Hemodialysis arteriovenous synthetic grafts (AVG provide high volumetric blood flow rates shortly after surgical placement. However, stenosis often develops at the vein-graft anastomosis contributing to thrombosis and early graft failure. Two novel fusion proteins, ANV-6L15 and TAP-ANV, inhibit the tissue factor/factor VIIa coagulation complex and the factor Xa/factor Va complex, respectively. Each inhibitor domain is fused to an annexin V domain that targets the inhibitor activity to sites of vascular injury to locally inhibit thrombosis. This study's objective was to determine if these antithrombotic proteins are safe and effective in inhibiting AVG stenosis.A bolus of either TAP-ANV or ANV-6L15 fusion protein was administered intravenously immediately prior to surgical placement of a synthetic graft between the external jugular vein and common carotid artery in a porcine model. At surgery, the vein and artery were irrigated with the anti-thrombotic fusion protein. Control animals received intravenous heparin. At 4 weeks, MRI was performed to evaluate graft patency, the pigs were then euthanized and grafts and attached vessels were explanted for histomorphometric assessment of neointimal hyperplasia at the vein-graft anastomosis. Blood was collected at surgery, immediately after surgery and at euthanasia for serum metabolic panels and coagulation chemistries.No acute thrombosis occurred in the control group or in either experimental group. No abnormal serum chemistries, activated clotting times or PT, PTT values were observed after treatment in experimental or control animals. However, at the vein-graft anastomosis, there was no difference between the control and experimental groups in cross-sectional lumen areas, as measured on MRI, and no difference in hyperplasia areas as determined by histomorphometry. These results suggest that local irrigation of TAP-ANV or ANV-6L15 intra-operatively was as effective in inhibiting acute graft thrombosis

  15. Inhibition of polyphenoloxidase by sulfite

    International Nuclear Information System (INIS)

    Sayavedra-Soto, L.A.; Montgomery, M.W.

    1986-01-01

    When polyphenoloxidase (PPO) was exposed to sulfite prior to substrate addition, inhibition was irreversible. Trials to regenerate PPO activity, using extensive dialysis, column chromatography, and addition of copper salts were not successful. Increased concentrations of sulfite and pH levels less than 5 enhanced the inhibition of PPO by sulfite. At pH 4, concentrations greater than 0.04 mg/mL completely inhibited 1000 units of PPO activity almost instantaneously. This suggested that the HSO 3 - molecule was the main component in the sulfite system inhibiting PPO. Column chromatography, extensive dialysis, and gel electrophoresis did not demonstrate 35 SO 2 bound to purified pear PPO protein. Formation of extra protein bands of sulfite inhibited purified pear PPO fractions on gel electrophoresis was demonstrated. This and other evidence suggested that the major mode of direct irreversible inhibition of PPO was modification of the protein structure, with retention of its molecular unity

  16. In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

    Science.gov (United States)

    Poovitha, Sundar; Parani, Madasamy

    2016-07-18

    α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

  17. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

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    Tetsuro Ikegami

    2009-02-01

    Full Text Available Rift Valley fever virus (RVFV (genus Phlebovirus, family Bunyaviridae is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR-mediated eukaryotic initiation factor (eIF2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

  18. Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

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    Abraham Lin

    Full Text Available Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage, these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

  19. General and specific lipid-protein interactions in Na,K-ATPase.

    Science.gov (United States)

    Cornelius, F; Habeck, M; Kanai, R; Toyoshima, C; Karlish, S J D

    2015-09-01

    The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions." Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Genetic ablation or chemical inhibition of phosphatidylcholine transfer protein attenuates diet-induced hepatic glucose production.

    Science.gov (United States)

    Shishova, Ekaterina Y; Stoll, Janis M; Ersoy, Baran A; Shrestha, Sudeep; Scapa, Erez F; Li, Yingxia; Niepel, Michele W; Su, Ya; Jelicks, Linda A; Stahl, Gregory L; Glicksman, Marcie A; Gutierrez-Juarez, Roger; Cuny, Gregory D; Cohen, David E

    2011-08-01

    Phosphatidylcholine transfer protein (PC-TP, synonym StARD2) is a highly specific intracellular lipid binding protein that is enriched in liver. Coding region polymorphisms in both humans and mice appear to confer protection against measures of insulin resistance. The current study was designed to test the hypotheses that Pctp-/- mice are protected against diet-induced increases in hepatic glucose production and that small molecule inhibition of PC-TP recapitulates this phenotype. Pctp-/- and wildtype mice were subjected to high-fat feeding and rates of hepatic glucose production and glucose clearance were quantified by hyperinsulinemic euglycemic clamp studies and pyruvate tolerance tests. These studies revealed that high-fat diet-induced increases in hepatic glucose production were markedly attenuated in Pctp-/- mice. Small molecule inhibitors of PC-TP were synthesized and their potencies, as well as mechanism of inhibition, were characterized in vitro. An optimized inhibitor was administered to high-fat-fed mice and used to explore effects on insulin signaling in cell culture systems. Small molecule inhibitors bound PC-TP, displaced phosphatidylcholines from the lipid binding site, and increased the thermal stability of the protein. Administration of the optimized inhibitor to wildtype mice attenuated hepatic glucose production associated with high-fat feeding, but had no activity in Pctp-/- mice. Indicative of a mechanism for reducing glucose intolerance that is distinct from commonly utilized insulin-sensitizing agents, the inhibitor promoted insulin-independent phosphorylation of key insulin signaling molecules. These findings suggest PC-TP inhibition as a novel therapeutic strategy in the management of hepatic insulin resistance. Copyright © 2011 American Association for the Study of Liver Diseases.

  1. Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging

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    Matthew C. Zimmerman

    2017-10-01

    Full Text Available Methotrexate (MTX is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD. Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA and acetaldehyde (AA, known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.

  2. Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT.

    Science.gov (United States)

    van Leeuwen, Hans; Okuwaki, Mitsuru; Hong, Rui; Chakravarti, Debabrata; Nagata, Kyosuke; O'Hare, Peter

    2003-09-01

    Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

  3. Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus*

    Science.gov (United States)

    Soares da Costa, Tatiana P.; Tieu, William; Yap, Min Y.; Pendini, Nicole R.; Polyak, Steven W.; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D.; Wallace, John C.; Wilce, Matthew C. J.; Booker, Grant W.; Abell, Andrew D.

    2012-01-01

    There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (Ki 90 nm) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class. PMID:22437830

  4. Selective inhibition of biotin protein ligase from Staphylococcus aureus.

    Science.gov (United States)

    Soares da Costa, Tatiana P; Tieu, William; Yap, Min Y; Pendini, Nicole R; Polyak, Steven W; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D; Wallace, John C; Wilce, Matthew C J; Booker, Grant W; Abell, Andrew D

    2012-05-18

    There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.

  5. Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

    Science.gov (United States)

    Zhen, Zijun; Yang, Kaibin; Ye, Litong; You, Zhiyao; Chen, Rirong; Liu, Ying; He, Youjian

    2017-07-01

    Paclitaxel is not as effective for neuroblastoma as most of the front-line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel-associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy-associated proteins were assessed by western blot. Autophagy was induced and the autophagy-associated proteins LC3-I, LC3-II, Beclin 1, and thioredoxin-related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1-mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel-induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel-induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  6. Selective memory generalization by spatial patterning of protein synthesis.

    Science.gov (United States)

    O'Donnell, Cian; Sejnowski, Terrence J

    2014-04-16

    Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings, we proposed a two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

    Directory of Open Access Journals (Sweden)

    Jennifer H Stuart

    2016-12-01

    Full Text Available The type I interferon (IFN response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3 complex to the interferon stimulated response element (ISRE. Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

  8. Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis.

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    Jody Groenendyk

    Full Text Available Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function.We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER homeostasis, transient activation of the unfolded protein response (UPR pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA is sufficient to prevent cardiac fibrosis, and improved exercise tolerance.We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.

  9. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    Science.gov (United States)

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  10. Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

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    Hisatomi Keiko

    2012-06-01

    Full Text Available Abstract Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF. We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

  11. The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

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    Galo L. Mejia

    2016-07-01

    Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

  12. R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4.

    Science.gov (United States)

    Wobst, Ivonne; Ebert, Lisa; Birod, Kerstin; Wegner, Marthe-Susanna; Hoffmann, Marika; Thomas, Dominique; Angioni, Carlo; Parnham, Michael J; Steinhilber, Dieter; Tegeder, Irmgard; Geisslinger, Gerd; Grösch, Sabine

    2016-12-30

    R -flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S -flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E₂ (PGE₂) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R -flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA 2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R -flurbiprofen traps PGE₂ inside of the cells by inhibiting multidrug resistance-associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R -flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R -flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.

  13. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    International Nuclear Information System (INIS)

    Choi, Ji-Woong; Kim, Jae-Hwan; Cho, Sung-Chun; Ha, Moon-Kyung; Song, Kye-Yong; Youn, Hong-Duk; Park, Sang Chul

    2011-01-01

    Research highlights: → ALDH2 is an MDA-modified protein in old rat kidney tissues. → AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. → ALDH2 serves as a general transcriptional repressor by associating with HDACs. → MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  14. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Ji-Woong [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of); Kim, Jae-Hwan [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Cho, Sung-Chun; Ha, Moon-Kyung [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of); Song, Kye-Yong [Department of Pathology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} ALDH2 is an MDA-modified protein in old rat kidney tissues. {yields} AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. {yields} ALDH2 serves as a general transcriptional repressor by associating with HDACs. {yields} MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  15. A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

    Science.gov (United States)

    Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2014-01-01

    Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

  16. Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

    2016-07-08

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

  17. Pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures

    International Nuclear Information System (INIS)

    Pratt, B.L.; Takahashi, J.S.

    1988-01-01

    The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation. Physiological response and [32P]ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal. For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed. Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner. Pertussis toxin-induced blockade appeared to be noncompetitive. One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition. Pertussis toxin-catalyzed [32P]ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin. In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by [32P]NAD. Pertussis toxin pretreatment of pineal cells abolished [32P] radiolabeling of the 40K Mr G-protein in a dose-dependent manner. The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin. Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by [32P]NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells

  18. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

    Science.gov (United States)

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

    2015-01-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

  19. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

    Directory of Open Access Journals (Sweden)

    Pierre-Emmanuel Joubert

    2015-08-01

    Full Text Available Chikungunya virus (CHIKV, the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K and MAP kinase-activated protein kinase (MnKs activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

  20. S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

    Science.gov (United States)

    Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

    2016-11-01

    Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I.

    Science.gov (United States)

    Gori-Savellini, Gianni; Valentini, Melissa; Cusi, Maria Grazia

    2013-06-01

    Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71-79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.

  2. Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways

    Directory of Open Access Journals (Sweden)

    Yinghong Ji

    2016-11-01

    Full Text Available Background/Aims: Ultraviolet B (UVB irradiation can easily induce apoptosis in human lens epithelial cells (HLECs and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1 gene in UVB irradiation induced-apoptosis in HLECs. Methods: Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. Results: After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm was increased in HLECs. Further studies indicated that superoxide dismutase (SOD activity and total antioxidative (T-AOC level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA and lactate dehydrogenase (LDH were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were significantly decreased, but the concentration of interleukin-10 (IL-10 was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38, Jun amino-terminal kinases (JNK1/2, phospho-JNK1/2 (p-JNK1/2, calcium-sensing receptor (CasR, and Ca2+/calmodulin-dependent protein kinase II (CaMKII indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. Conclusion: These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment.

  3. Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways.

    Science.gov (United States)

    Ji, Yinghong; Rong, Xianfang; Li, Dan; Cai, Lei; Rao, Jun; Lu, Yi

    2016-01-01

    Ultraviolet B (UVB) irradiation can easily induce apoptosis in human lens epithelial cells (HLECs) and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1) gene in UVB irradiation induced-apoptosis in HLECs. Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS) production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm) was increased in HLECs. Further studies indicated that superoxide dismutase (SOD) activity and total antioxidative (T-AOC) level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly decreased, but the concentration of interleukin-10 (IL-10) was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38), Jun amino-terminal kinases (JNK1/2), phospho-JNK1/2 (p-JNK1/2), calcium-sensing receptor (CasR), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment. © 2016 The Author(s) Published by S. Karger AG, Basel.

  4. The Crc protein inhibits the production of polyhydroxyalkanoates in Pseudomonas putida under balanced carbon/nitrogen growth conditions.

    Science.gov (United States)

    La Rosa, Ruggero; de la Peña, Fernando; Prieto, María Axiliadora; Rojo, Fernando

    2014-01-01

    Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  5. A monocyte chemotaxis inhibiting factor in serum of HIV infected men shares epitopes with the HIV transmembrane protein gp41

    NARCIS (Netherlands)

    Tas, M.; Drexhage, H. A.; Goudsmit, J.

    1988-01-01

    This report describes that gp41, the transmembranous envelope protein of HIV, is able to inhibit monocyte chemotaxis (measured as FMLP-induced polarization). To study the presence of such immunosuppressive HIV env proteins in the circulation of HIV-infected men, fractions were prepared from serum

  6. Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

    Science.gov (United States)

    Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

    2015-04-01

    Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

  7. BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast.

    Science.gov (United States)

    Juhásová, Barbora; Mentel, Marek; Bhatia-Kiššová, Ingrid; Zeman, Igor; Kolarov, Jordan; Forte, Michael; Polčic, Peter

    2011-09-02

    Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Inhibition of fatty acid binding proteins elevates brain anandamide levels and produces analgesia.

    Directory of Open Access Journals (Sweden)

    Martin Kaczocha

    Full Text Available The endocannabinoid anandamide (AEA is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH. Fatty acid binding proteins (FABPs are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1 and peroxisome proliferator-activated receptor alpha (PPARα and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

  9. A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    Full Text Available For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III based on the protein sequence and structure. For Rift Valley fever virus (RVFV, the glycoprotein Gc (Class II fusion protein mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus, Class II (Andes virus, or Class III (vesicular stomatitis virus fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

  10. The kampo medicine Daikenchuto inhibits peritoneal fibrosis in mice.

    Science.gov (United States)

    Kitamura, Mineaki; Nishino, Tomoya; Obata, Yoko; Oka, Satoru; Abe, Shinichi; Muta, Kumiko; Ozono, Yoshiyuki; Koji, Takehiko; Kohno, Shigeru

    2015-01-01

    Long-term peritoneal dialysis therapy causes inflammation and histological changes in the peritoneal membrane. Inflammation generally activates fibroblasts and results in fibroblast-myofibroblast differentiation. Heat-shock protein 47 (HSP 47), a collagen-specific molecular chaperone, is localized in myofibroblasts and is involved in the progression of peritoneal fibrosis. Daikenchuto (DKT), a Kampo medicine, is used to prevent postoperative colon adhesion. It inhibits inflammation and HSP 47 expression in the gastrointestinal tract. We examined the effect of DKT on chlorhexidine gluconate (CG)-induced peritoneal fibrosis in mice injected with 0.1% CG dissolved in 15% ethanol. DKT was dissolved in the drinking water. Histological changes were assessed using Masson trichrome staining. Cells expressing α-smooth muscle actin (α-SMA), HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 were examined immunohistochemically. Compared with the control group, the peritoneal tissues of the CG group were markedly thickened, and the number of cells expressing α-SMA, HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 was significantly increased. However, these changes were inhibited in the DKT-treated group. These results indicate that DKT can prevent peritoneal fibrosis by inhibiting inflammation and HSP 47 expression.

  11. Glioblastoma Inhibition by Cell Surface Immunoglobulin Protein EWI-2, In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana V. Kolesnikova

    2009-01-01

    Full Text Available EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP, which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.

  12. RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair

    Directory of Open Access Journals (Sweden)

    Mohammad A.M. Ali

    2018-01-01

    Full Text Available Summary: Ring1-YY1-binding protein (RYBP is a member of the non-canonical polycomb repressive complex 1 (PRC1, and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs, we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding. : Ali et al. find that RYBP binds K63-linked ubiquitin chains and is removed from DNA damage sites. This K63-ubiquitin binding allows RYBP to hinder the recruitment of BRCA1 and Rad51 to DNA double-strand breaks, thus inhibiting homologous recombination repair. Accordingly, cancer cells expressing high RYBP are more sensitive to DNA-damaging therapies. Keywords: DNA damage response, homologous recombination, ubiquitylation, RYBP, polycomb proteins, double-strand break repair, chromatin, histone modification

  13. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    Science.gov (United States)

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-08-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

  14. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Science.gov (United States)

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  15. Molecular cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum, and their relation to increased resistance to two fungal pathogens

    Science.gov (United States)

    Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). Inhibiting by PGIPs directly reduces potential PG activity in specific plant pathogenic fungi, reducing their aggressiveness. Here, we isolated and functionally chara...

  16. Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function.

    Science.gov (United States)

    Chauhan, Jay; Chen, Shen-En; Fenstermacher, Katherine J; Naser-Tavakolian, Aurash; Reingewertz, Tali; Salmo, Rosene; Lee, Christian; Williams, Emori; Raje, Mithun; Sundberg, Eric; DeStefano, Jeffrey J; Freire, Ernesto; Fletcher, Steven

    2015-11-01

    Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Ascorbate attenuates pulmonary emphysema by inhibiting tobacco smoke and Rtp801-triggered lung protein modification and proteolysis.

    Science.gov (United States)

    Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R; Stuehr, Dennis J; Panda, Koustubh

    2016-07-19

    Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.

  18. Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

    Science.gov (United States)

    Calder, Michele D; Watson, Patricia H; Watson, Andrew J

    2011-11-01

    During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

  19. Inhibition of the NAD-dependent protein deacetylase SIRT2 induces granulocytic differentiation in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sunami

    Full Text Available Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

  20. Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo.

    Science.gov (United States)

    Goodman, Craig A; McNally, Rachel M; Hoffmann, F Michael; Hornberger, Troy A

    2013-11-01

    Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.

  1. Sources of dietary protein and risk of hypertension in a general Dutch population

    NARCIS (Netherlands)

    Altorf-van der Kuil, W.; Engberink, M.F.; Geleijnse, J.M.; Boer, J.M.A.; Verschuren, W.M.M.

    2012-01-01

    Evidence suggests a small beneficial effect of dietary protein on blood pressure (BP), especially for plant protein. We examined the relationship between several types of dietary protein (total, plant, animal, dairy, meat and grain) and the risk of hypertension in a general population of 3588 Dutch

  2. Inhibition of the neutrophil oxidative burst by sphingoid long-chain bases: role of protein kinase C in the activation of the burst

    International Nuclear Information System (INIS)

    Wilson, E.; Olcott, M.C.; Bell, R.M.; Merrill, A.H.; Lambeth, J.D.

    1986-01-01

    The neutrophil oxidative burst is triggered by a variety of both particulate (opsonized zymosan) and soluble agonists [formylmethionylleucylphenylalanine (FMLP), arachidonate, short-chained diacylglycerols (DAG) and phorbol myristate acetate (PMA)]. The authors show that the long-chain lipid bases sphinganine and sphingosine block activation of the burst in human neutrophils. Inhibition is reversible, does not alter cell viability, and does not affect phagocytosis. The inhibition affects the activation mechanism rather than the NADPH-oxidase enzyme. The structural requirements for inhibition include a hydrophobic carbon chain and an amino-containing headgroup, and the naturally occurring erythro sphinganine was more potent than the threo isomer. Activation of the oxidative burst by a variety of agonists was blocked by the same concentration of sphinganine indicating a common inhibited step. The authors suggest that the common step is protein kinase C, as evidenced by the following: 1) long-chain bases inhibit PKC in a micelle reconstituted system, 2) PMA-induced phophorylation is inhibited by sphinganine, and 3) sphinganine competes with ( 3 H)-phorbol dibutyrate for its cytosolic receptor (i.e. protein kinase C). The authors suggest that sphingoid long-chain bases play a role in the cellular regulations

  3. Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state and upon deformylation inhibition.

    Science.gov (United States)

    Bienvenut, Willy V; Giglione, Carmela; Meinnel, Thierry

    2015-07-01

    A proteome wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of actinonin, an antibiotic specifically inhibiting peptide deformylase (PDF). A strategy and tool suite (SILProNaQ) was employed to provide large-scale quantitation of N-terminal modifications. In control conditions, more than 1000 unique N-termini were identified with 56% showing initiator methionine removal. Additional modifications corresponded to partial or complete Nα-acetylation (10%) and N-formyl retention (5%). Among the proteins undergoing these N-terminal modifications, 140 unique N-termini from translocated membrane proteins were highlighted. The very early time-course impact of actinonin was followed after addition of bacteriostatic concentrations of the drug. Under these conditions, 26% of all proteins did not undergo deformylation any longer after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation ratio measured on individual proteins increased with the same trend. Upon early PDF inhibition, two major categories of proteins retained their N-formyl group: a large number of inner membrane proteins and many proteins involved in protein synthesis including factors assisting the nascent chains in early cotranslational events. All MS data have been deposited in the ProteomeXchange with identifiers PXD001979, PXD002012 and PXD001983 (http://proteomecentral.proteomexchange.org/dataset/PXD001979, http://proteomecentral.proteomexchange.org/dataset/PXD002012 and http://proteomecentral.proteomexchange.org/dataset/PXD001983). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-05-01

    Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4

    Directory of Open Access Journals (Sweden)

    Ivonne Wobst

    2016-12-01

    Full Text Available R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2 in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance–associated protein 4 (MRP4, ABCC4, which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.

  6. Red cell autoantibodies characterized by competitive inhibition of iodine 125 Rh alloantibody binding and by immunoprecipitation of membrane proteins

    International Nuclear Information System (INIS)

    Pierce, S.W.; Victoria, E.J.; Masouredis, S.P.

    1990-01-01

    The relationship between determinants recognized by warm-type immunoglobulin G red cell autoantibodies and the Rh antigens was characterized by autoantibody competitive inhibition of iodine 125 Rh alloantibody binding and autoantibody immunoprecipitation of iodine 125 red blood cell membrane proteins. The majority of blood donor autoantibody recognized epitopes that are closely related to Rh antigens as determined by competitive inhibition studies. Eighteen of 20 (90%) autoantibodies inhibited anti-Rh(c) binding, 15 inhibited anti-Rh(E), 5 inhibited anti-Rh(D), and only 2 failed to inhibit any of the three Rh alloantibodies tested. Autoantibodies that inhibited anti-Rh(D) also inhibited anti-Rh(c) and anti-Rh(E) and all those that inhibited anti-Rh(E) also inhibited anti-Rh(c). Autoantibodies that inhibited all three Rh alloantibodies immunoprecipitated 30 kd membrane polypeptides, as did two of the three autoantibodies that inhibited only anti-Rh(c) and anti-Rh(E). One autoantibody in this group and two autoantibodies that inhibited only anti-Rh(c), as well as an autoantibody that did not inhibit any of the Rh alloantibodies, immunoprecipitated only a single membrane polypeptide identified as band 3. The majority of normal donor red blood cell autoantibodies inhibited the binding of Rh alloantibodies, which indicates that they either bound to the Rh polypeptides or to epitopes on band 3 that were closely associated with the Rh complex

  7. Nuclear protein IκB-ζ inhibits the activity of STAT3

    International Nuclear Information System (INIS)

    Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

    2009-01-01

    STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

  8. Laticifer proteins from Plumeria pudica inhibit the inflammatory and nociceptive responses by decreasing the action of inflammatory mediators and pro-inflammatory cytokines

    Directory of Open Access Journals (Sweden)

    Heliana B. Fernandes

    Full Text Available AbstractSome publications have described the pharmacological properties of latices proteins. Thus, in the present study proteins from Plumeria pudica Jacq., Apocynaceae, latex were evaluated for anti-inflammatory and antinociceptive activities. Obtained data showed that an intraperitoneal administration of different doses of latex was able to reduce the paw edema induced by carrageenan in a dose-dependent manner (better dose 40 mg/kg; 72.7% inhibition at 3rd and 78.7% at 4th hour and the edema induced by dextran (40 mg/kg; 51.5% inhibition at 30 min and 93.0% at 1st hour. Inhibition of edema induced by carrageenan was accompanied by a reduction of myeloperoxidase activity. Pre-treating animals with latex (40 mg/kg also inhibited the paw edema induced by histamine, serotonin, bradykinin, prostaglandin E2, compound 48/80. Additionally, the latex (40 mg/kg reduced the leukocyte peritoneal migration induced by carrageenan and this event was followed by reduction of IL-1β and TNF-α in peritoneal fluid. The latex-treatment (40 mg/kg reduced the animal abdominal constrictions induced by acetic acid and the first phase on paw licking model induced by formalin. When latex was treated with heat (at 100 °C for 30 min, anti-edematogenic and myeloperoxidase activities were significantly reduced, indicating the involvement of heat-sensitive proteins on anti-inflammatory effect. Our results evidence that latex fluids are a source of proteins with pharmacological properties.

  9. Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

    Science.gov (United States)

    Kalous, Kelsey S; Wynia-Smith, Sarah L; Olp, Michael D; Smith, Brian C

    2016-12-02

    The sirtuin family of proteins catalyze the NAD + -dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD + and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn 2+ release from the conserved sirtuin Zn 2+ -tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn 2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD + and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn 2+ , consistent with S-nitrosation of the Zn 2+ -tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  11. Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

    International Nuclear Information System (INIS)

    Pirsel, M.; Slezarikova, V.

    1977-01-01

    A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

  12. Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells.

    Science.gov (United States)

    Santo-Domingo, Jaime; Chareyron, Isabelle; Broenimann, Charlotte; Lassueur, Steve; Wiederkehr, Andreas

    2017-08-15

    Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed

  13. End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1.

    Science.gov (United States)

    Lescasse, Rachel; Pobiega, Sabrina; Callebaut, Isabelle; Marcand, Stéphane

    2013-03-20

    In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.

  14. Stimulation of a Cd-binding protein, and inhibition of the vitamin D-dependent calcium-binding protein, by zinc or cadmium in organ-cultured embryonic chick duodenum

    International Nuclear Information System (INIS)

    Corradino, R.A.; Fullmer, C.S.

    1980-01-01

    Embryonic chick duodenum maintained in organ culture responds to 1 α,25-dihydroxy vitamin D 3 in the culture medium by de novo synthesis of a specific calcium-binding protein (CaBP). The addition of Cd 2+ (3-5 x 10 -5 M) or Zn 2+ (10 -5 -10 -4 M) to the medium inhibited CaBP, but stimulated biosynthesis of a Cd-binding protein (CdBP). CdBP in duodenal homogenate supernatants was assessed in two ways: first, by its 109 Cd-binding activity ( 109 CdBA) using a competitive ion exchange procedure; and, second, by the extent of [ 35 S]-cystine incorporation into a specific peak or band after gel filtration or analytical polyacrylamide disc gel electrophoresis, respectively. Regardless of whether cadmium- or zinc-stimulated, the 35 S-labeled CdBP - the only protein significantly labeled under the conditions employed - migrated identically upon gel filtration and electrophoresis, and comigrated with purified chick liver Cd-metallothionein. Neither actinomycin D nor α-amanitin, in concentrations sufficient to severely inhibit CaBP, significantly reduced CdBP production. However, cycloheximide did inhibit either Cd 2+ - or Zn 2+ -stimulated CdBP by about 50% at an inhibitor concentration which abolished CaBP. The inhibitor studies, coupled with the observations of extensive incorporation of [ 35 S]cystine into CdBP, suggest that the metals stimulated biosynthesis by a mechanism operating at the translational level. The organ-cultured duodenum seems well suited for studies of the regulation of CdBP biosynthesis especially since it responds predictably to the steroid hormone, 1α,25-dihydroxy vitamin D 3 , in the induction of another specific protein, CaBP, at the transcriptional level. The biosynthesis of CaBP thus may serve as a convenient control in studies of CdBP production under various experimental conditions

  15. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    Science.gov (United States)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  16. Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

    Science.gov (United States)

    Hoyt, Laura R.; Ather, Jennifer L.; Randall, Matthew J.; DePuccio, Daniel P.; Landry, Christopher C.; Wewers, Mark D.; Gavrilin, Mikhail A.; Poynter, Matthew E.

    2016-01-01

    Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished ASC speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of GABAA receptor activation or NMDA receptor inhibition, but was associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC, were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols. PMID:27421477

  17. The cytotoxic effects of regorafenib in combination with protein kinase D inhibition in human colorectal cancer cells

    Science.gov (United States)

    Wei, Ning; Chu, Edward; Wu, Shao-yu; Wipf, Peter; Schmitz, John C.

    2015-01-01

    Metastatic colorectal cancer (mCRC) remains a major public health problem, and diagnosis of metastatic disease is usually associated with poor prognosis. The multi-kinase inhibitor regorafenib was approved in 2013 in the U.S. for the treatment of mCRC patients who progressed after standard therapies. However, the clinical efficacy of regorafenib is quite limited. One potential strategy to improve mCRC therapy is to combine agents that target key cellular signaling pathways, which may lead to synergistic enhancement of antitumor efficacy and overcome cellular drug resistance. Protein kinase D (PKD), a family of serine/threonine kinases, mediates key signaling pathways implicated in multiple cellular processes. Herein, we evaluated the combination of regorafenib with a PKD inhibitor in several human CRC cells. Using the Chou-Talalay model, the combination index values for this combination treatment demonstrated synergistic effects on inhibition of cell proliferation and clonal formation. This drug combination resulted in induction of apoptosis as determined by flow cytometry, increased PARP cleavage, and decreased activation of the anti-apoptotic protein HSP27. This combination also yielded enhanced inhibition of ERK, AKT, and NF-κB signaling. Taken together, PKD inhibition in combination with regorafenib appears to be a promising strategy for the treatment of mCRC. PMID:25544765

  18. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    Science.gov (United States)

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

  19. Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

    International Nuclear Information System (INIS)

    Kim, Seong K.; Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

    2011-01-01

    The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: → We examine the functional domains of IR2P that mediates negative regulation. → IR2P inhibits at the transcriptional level. → DNA-binding mutant or TFIIB-binding mutant fails to inhibit. → C-terminal aa 707 to 1116 are required for full inhibition. → Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

  20. A general theory of non-equilibrium dynamics of lipid-protein fluid membranes

    DEFF Research Database (Denmark)

    Lomholt, Michael Andersen; Hansen, Per Lyngs; Miao, L.

    2005-01-01

    We present a general and systematic theory of non-equilibrium dynamics of multi-component fluid membranes, in general, and membranes containing transmembrane proteins, in particular. Developed based on a minimal number of principles of statistical physics and designed to be a meso...

  1. Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins

    International Nuclear Information System (INIS)

    Krawczyk, P. M.; Stap, C.; Van Oven, C.; Hoebe, R.; Aten, J. A.

    2006-01-01

    For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains. (authors)

  2. Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-β2 in Human TM Cells.

    Science.gov (United States)

    Mody, Avani A; Wordinger, Robert J; Clark, Abbot F

    2017-02-01

    Increased expression of TGF-β2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-β2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-β2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-β2 induction of ECM proteins in TM cells. Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-β2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-β2-induced expression of fibronectin and PAI-1 in TM cells (P protein 4 induced ID1 and ID3 expression suppresses TGF-β2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-β2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

  3. Alzheimer's Disease Brain-Derived Amyloid-{beta}-Mediated Inhibition of LTP In Vivo Is Prevented by Immunotargeting Cellular Prion Protein.

    LENUS (Irish Health Repository)

    Barry, Andrew E

    2011-05-18

    Synthetic amyloid-β protein (Aβ) oligomers bind with high affinity to cellular prion protein (PrP(C)), but the role of this interaction in mediating the disruption of synaptic plasticity by such soluble Aβ in vitro is controversial. Here we report that intracerebroventricular injection of Aβ-containing aqueous extracts of Alzheimer\\'s disease (AD) brain robustly inhibits long-term potentiation (LTP) without significantly affecting baseline excitatory synaptic transmission in the rat hippocampus in vivo. Moreover, the disruption of LTP was abrogated by immunodepletion of Aβ. Importantly, intracerebroventricular administration of antigen-binding antibody fragment D13, directed to a putative Aβ-binding site on PrP(C), prevented the inhibition of LTP by AD brain-derived Aβ. In contrast, R1, a Fab directed to the C terminus of PrP(C), a region not implicated in binding of Aβ, did not significantly affect the Aβ-mediated inhibition of LTP. These data support the pathophysiological significance of SDS-stable Aβ dimer and the role of PrP(C) in mediating synaptic plasticity disruption by soluble Aβ.

  4. Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Xie, Ji-Sheng [Youjiang Medical College for Nationalities, Guangxi 533000 (China); Meng, Xin; Guan, Yifu [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Wang, Hua-Qin, E-mail: wanghq_doctor@hotmail.com [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)

    2010-01-01

    Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2{alpha} inhibitor, or overexpression of dominant negative mutants of PERK or eIF2{alpha}, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2{alpha} branch of UPR in RES-induced inhibition of cell proliferation.

  5. Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

    International Nuclear Information System (INIS)

    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang; Xie, Ji-Sheng; Meng, Xin; Guan, Yifu; Wang, Hua-Qin

    2010-01-01

    Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2α (eIF2α), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2α inhibitor, or overexpression of dominant negative mutants of PERK or eIF2α, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2α branch of UPR in RES-induced inhibition of cell proliferation.

  6. Inhibition of Vascular Smooth Muscle Growth via Signaling Crosstalk between AMP-Activated Protein Kinase and cAMP-Dependent Protein Kinase

    Directory of Open Access Journals (Sweden)

    Joshua Daniel Stone

    2012-10-01

    Full Text Available Abnormal vascular smooth muscle (VSM growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK and cAMP-dependent protein kinase (PKA. Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remains unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells, the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSM cell migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashions. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.

  7. Dual orexin receptor antagonist 12 inhibits expression of proteins in neurons and glia implicated in peripheral and central sensitization.

    Science.gov (United States)

    Cady, R J; Denson, J E; Sullivan, L Q; Durham, P L

    2014-06-06

    Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX₁R) and receptor 2 (OX₂R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX₁R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central

  8. Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

    Science.gov (United States)

    Mansilla, Sylvia; Rojas, Marta; Bataller, Marc; Priebe, Waldemar; Portugal, José

    2007-04-01

    Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC(75) for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

  9. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis.

    Science.gov (United States)

    Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

    2013-01-01

    Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose) polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy.

  10. Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications.

    Directory of Open Access Journals (Sweden)

    Arantza Soler-Cantero

    Full Text Available Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine-protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu(++-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters. This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

  11. Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

    Directory of Open Access Journals (Sweden)

    Robert M Gill

    Full Text Available Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-/hydrogen peroxide (H2O2 release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM. This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

  12. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

    Directory of Open Access Journals (Sweden)

    Ramón A. Lorca

    2011-01-01

    Full Text Available Although the physiological function of the cellular prion protein (PrPC remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

  13. Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

    International Nuclear Information System (INIS)

    Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

    1989-01-01

    The effects of three different concentrations (about 20, 100 and 1000 μM) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, 14 C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively. (author)

  14. Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

    Energy Technology Data Exchange (ETDEWEB)

    Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

    1989-01-01

    The effects of three different concentrations (about 20, 100 and 1000 ..mu..M) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, /sup 14/C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively.

  15. Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

    International Nuclear Information System (INIS)

    Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J.

    1996-01-01

    The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6- 3 H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

  16. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    Energy Technology Data Exchange (ETDEWEB)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-08-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: (/sup 3/H)thymidine incorporation decreased by 80% and (/sup 14/C)leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of (/sup 3/H)histidine/(/sup 14/C)leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

  17. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    International Nuclear Information System (INIS)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-01-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: [ 3 H]thymidine incorporation decreased by 80% and [ 14 C]leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of [ 3 H]histidine/[ 14 C]leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine

  18. Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex

    International Nuclear Information System (INIS)

    Hostetler, K.Y.; Gardner, M.F.; Giordano, J.R.

    1986-01-01

    Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1- 14 C]oleoylphosphatidylcholine by purified phospholipase A 1 with IC 50 values of 7-11 μg. The inhibition is abolished by preincubation with trypsin at 37 0 C, but preincubation with trypsin at 4 0 C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither rho-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A 1 . In contrast to rat liver, which has two major isoenzymes of acid phospholipase A 1 , kidney cortex has only one isoenzyme of lysosomal phospholipase A 1

  19. Extracts Obtained from Pterocarpus angolensis DC and Ziziphus mucronata Exhibit Antiplasmodial Activity and Inhibit Heat Shock Protein 70 (Hsp70 Function

    Directory of Open Access Journals (Sweden)

    Tawanda Zininga

    2017-07-01

    Full Text Available Malaria parasites are increasingly becoming resistant to currently used antimalarial therapies, therefore there is an urgent need to expand the arsenal of alternative antimalarial drugs. In addition, it is also important to identify novel antimalarial drug targets. In the current study, extracts of two plants, Pterocarpus angolensis and Ziziphus mucronata were obtained and their antimalarial functions were investigated. Furthermore, we explored the capability of the extracts to inhibit Plasmodium falciparum heat shock protein 70 (Hsp70 function. Heat shock protein 70 (Hsp70 are molecular chaperones whose function is to facilitate protein folding. Plasmodium falciparum the main agent of malaria, expresses two cytosol-localized Hsp70s: PfHsp70-1 and PfHsp70-z. The PfHsp70-z has been reported to be essential for parasite survival, while inhibition of PfHsp70-1 function leads to parasite death. Hence both PfHsp70-1 and PfHsp70-z are potential antimalarial drug targets. Extracts of P. angolensis and Z. mucronata inhibited the basal ATPase and chaperone functions of the two parasite Hsp70s. Furthermore, fractions of P. angolensis and Z. mucronata inhibited P. falciparum 3D7 parasite growth in vitro. The extracts obtained in the current study exhibited antiplasmodial activity as they killed P. falciparum parasites maintained in vitro. In addition, the findings further suggest that some of the compounds in P. angolensis and Z. mucronata may target parasite Hsp70 function.

  20. Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex

    Energy Technology Data Exchange (ETDEWEB)

    Hostetler, K.Y.; Gardner, M.F.; Giordano, J.R.

    1986-10-21

    Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di(1-/sup 14/C)oleoylphosphatidylcholine by purified phospholipase A/sub 1/ with IC/sub 50/ values of 7-11 ..mu..g. The inhibition is abolished by preincubation with trypsin at 37/sup 0/C, but preincubation with trypsin at 4/sup 0/C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither rho-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A/sub 1/. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A/sub 1/, kidney cortex has only one isoenzyme of lysosomal phospholipase A/sub 1/.

  1. Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

    Science.gov (United States)

    Hansen, T N; Carpenter, J F

    1993-01-01

    Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP. Images FIGURE 8 PMID:7690257

  2. Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2

    NARCIS (Netherlands)

    Zanden, J.J. van; Wortelboer, H.M.; Bijlsma, S.; Punt, A.; Usta, M.; Bladeren, P.J.V.; Rietjens, I.M.C.M.; Cnubben, N.H.P.

    2005-01-01

    In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated

  3. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

    International Nuclear Information System (INIS)

    Apostolou, Andria; Shen Yuxian; Liang Yan; Luo Jun; Fang Shengyun

    2008-01-01

    The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR

  4. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing; Zhu, Jian-Kang

    2010-01-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host's essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  5. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

    Science.gov (United States)

    Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

    2012-10-01

    PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

  6. NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

    Science.gov (United States)

    Rapino, F; Abhari, B A; Jung, M; Fulda, S

    2015-03-12

    Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

  7. How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations

    DEFF Research Database (Denmark)

    Aijanen, T.; Koivuniemi, A.; Javanainen, M.

    2014-01-01

    Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity...... and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics...... simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site...

  8. [The role of balanced low-protein diet in inhibition of predialysis chronic kidney disease progression in patients with systemic diseases].

    Science.gov (United States)

    Milovanov, Iu S; Lysenko, L V; Milovanova, L Iu; Dobrosmyslov, I A

    2009-01-01

    To evaluate the effects of low-protein diet (LPD) balanced by addition of highly energetic mix and essential keto/amino acids on inhibition of renal failure in patients with systemic diseases with predialysis stages of chronic disease of the kidney (CDK). Forty six patients with stage III--IV of CDK in systemic diseases (33 SLE patients and 13 with systemic vasculitis) were randomized into three groups. Group 1 consisted of 18 patients with CDK (10 with stage III and 8 with stage IV). They received LPD (0.6 g/kg/day) with addition of essential keto/amino acids for 24-48 months. Group 2 of 18 CDK patients with the same stages received the same diet but greater amount of vegetable protein (highly purified soya protein) to 0.3 g/kg/day in highly energetic nutrient mixture. Group 3--10 CDK patients (7 with stage III and 3 with stage IV) received free diet. Group 1 and 2 patients received LPD irrespective of the nutrient status assessed basing on anthropometric and other data. Protein consumption and caloric value were estimated by 3-day food diary. Before diet therapy, out of 46 examinees nutrient status was abnormal in 45.7% patients. Both variants of LPD were well tolerated and nutrient status was corrected while the rate of nutritive disorders in group 3 increased 1.5-fold (from 40 to 60%) with progression of renal failure. Intake of LPD diet for at least a year reduced glomerular filtration rate inhibition, especially in addition of highly energetic mixture. Early (predialysis) restriction of diet protein (0.6 g/kg/day) with addition of highly energetic mixture and essential keto/amino acids improves a nutritive status of CDK patients and inhibits GFR decline.

  9. Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.

    Science.gov (United States)

    John, Ulrik P; Polotnianka, Renatam M; Sivakumaran, Kailayapillai A; Chew, Orinda; Mackin, Leanne; Kuiper, Micheal J; Talbot, Jonathan P; Nugent, Gregory D; Mautord, Julie; Schrauf, Gustavo E; Spangenberg, German C

    2009-04-01

    Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

  10. Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

    Science.gov (United States)

    Calpe, Silvia; Wagner, Koen; El Khattabi, Mohamed; Rutten, Lucy; Zimberlin, Cheryl; Dolk, Edward; Verrips, C Theo; Medema, Jan Paul; Spits, Hergen; Krishnadath, Kausilia K

    2015-11-01

    Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies. ©2015 American Association for Cancer Research.

  11. The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Famin O.

    2003-03-01

    Full Text Available Ferriprotoporphyrin IX (FPIX is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleoside monophosphate kinase and pyrimidine 5'- nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-α, phoshoglycerate kinase, glyceraldehyde 3- phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6- phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

  12. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    International Nuclear Information System (INIS)

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-01-01

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [ 3 H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [ 3 H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  13. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    Science.gov (United States)

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

    Science.gov (United States)

    Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

    2005-11-01

    Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in

  15. Control of Excitation/Inhibition Balance in a Hippocampal Circuit by Calcium Sensor Protein Regulation of Presynaptic Calcium Channels.

    Science.gov (United States)

    Nanou, Evanthia; Lee, Amy; Catterall, William A

    2018-05-02

    Activity-dependent regulation controls the balance of synaptic excitation to inhibition in neural circuits, and disruption of this regulation impairs learning and memory and causes many neurological disorders. The molecular mechanisms underlying short-term synaptic plasticity are incompletely understood, and their role in inhibitory synapses remains uncertain. Here we show that regulation of voltage-gated calcium (Ca 2+ ) channel type 2.1 (Ca V 2.1) by neuronal Ca 2+ sensor (CaS) proteins controls synaptic plasticity and excitation/inhibition balance in a hippocampal circuit. Prevention of CaS protein regulation by introducing the IM-AA mutation in Ca V 2.1 channels in male and female mice impairs short-term synaptic facilitation at excitatory synapses of CA3 pyramidal neurons onto parvalbumin (PV)-expressing basket cells. In sharp contrast, the IM-AA mutation abolishes rapid synaptic depression in the inhibitory synapses of PV basket cells onto CA1 pyramidal neurons. These results show that CaS protein regulation of facilitation and inactivation of Ca V 2.1 channels controls the direction of short-term plasticity at these two synapses. Deletion of the CaS protein CaBP1/caldendrin also blocks rapid depression at PV-CA1 synapses, implicating its upregulation of inactivation of Ca V 2.1 channels in control of short-term synaptic plasticity at this inhibitory synapse. Studies of local-circuit function revealed reduced inhibition of CA1 pyramidal neurons by the disynaptic pathway from CA3 pyramidal cells via PV basket cells and greatly increased excitation/inhibition ratio of the direct excitatory input versus indirect inhibitory input from CA3 pyramidal neurons to CA1 pyramidal neurons. This striking defect in local-circuit function may contribute to the dramatic impairment of spatial learning and memory in IM-AA mice. SIGNIFICANCE STATEMENT Many forms of short-term synaptic plasticity in neuronal circuits rely on regulation of presynaptic voltage-gated Ca 2+ (Ca V

  16. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

    Science.gov (United States)

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

    2016-01-01

    Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function

  17. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

    Directory of Open Access Journals (Sweden)

    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  18. Antihelminthic drug niclosamide inhibits CIP2A and reactivates tumor suppressor protein phosphatase 2A in non-small cell lung cancer cells.

    Science.gov (United States)

    Kim, Myeong-Ok; Choe, Min Ho; Yoon, Yi Na; Ahn, Jiyeon; Yoo, Minjin; Jung, Kwan-Young; An, Sungkwan; Hwang, Sang-Gu; Oh, Jeong Su; Kim, Jae-Sung

    2017-11-15

    Protein phosphatase 2A (PP2A) is a critical tumor suppressor complex responsible for the inactivation of various oncogenes. Recently, PP2A reactivation has emerged asan anticancer strategy. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A, is upregulated in many cancer cells, including non-small cell lung cancer (NSCLC) cells. We demonstrated that the antihelminthic drug niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A in NSCLC cells. We performed a drug-repurposing screen and identified niclosamide asa CIP2A suppressor in NSCLC cells. Niclosamide inhibited cell proliferation, colony formation, and tumor sphere formation, and induced mitochondrial dysfunction through increased mitochondrial ROS production in NSCLC cells; however, these effects were rescued by CIP2A overexpression, which indicated that the antitumor activity of niclosamide was dependent on CIP2A. We found that niclosamide increased PP2A activity through CIP2A inhibition, which reduced the phosphorylation of several oncogenic proteins. Moreover, we found that a niclosamide analog inhibited CIP2A expression and increased PP2A activity in several types of NSCLC cells. Finally, we showed that other well-known PP2A activators, including forskolin and FTY720, did not inhibit CIP2A and that their activities were not dependent on CIP2A. Collectively, our data suggested that niclosamide effectively suppressed CIP2A expression and subsequently activated PP2A in NSCLC cells. This provided strong evidence for the potential use of niclosamide asa PP2A-activating drug in the clinical treatment of NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing

    2010-05-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  20. Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

    Science.gov (United States)

    Correa, Agustín; Pacheco, Sabino; Mechaly, Ariel E; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M; Pecorari, Frédéric

    2014-01-01

    Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

  1. Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins.

    Directory of Open Access Journals (Sweden)

    Agustín Correa

    Full Text Available Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM and CelD (Ki = 95 and 111 nM, high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

  2. Evaluations of in vitro metabolism, drug-drug interactions mediated by reversible and time-dependent inhibition of CYPs, and plasma protein binding of MMB4 DMS.

    Science.gov (United States)

    Hong, S Peter; Lusiak, Bozena D; Burback, Brian L; Johnson, Jerry D

    2013-01-01

    1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When (14)C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.

  3. Protein interactions in genome maintenance as novel antibacterial targets.

    Directory of Open Access Journals (Sweden)

    Aimee H Marceau

    Full Text Available Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  4. Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

    International Nuclear Information System (INIS)

    Ransjoe, M.; Lerner, U.H.

    1990-01-01

    Calcitonin is a well known inhibitor of osteoclastic bone resortion, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called ''escape from inhibition'' phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45 Ca. Two proteon kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45 Ca release in 120-h cultures at a concentration of 10 nmul/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any ''escape from inhibition''. This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E 2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption. (author)

  5. Evaluating the Role of Multidrug Resistance Protein 3 (MDR3) Inhibition in Predicting Drug-Induced Liver Injury Using 125 Pharmaceuticals.

    Science.gov (United States)

    Aleo, Michael D; Shah, Falgun; He, Kan; Bonin, Paul D; Rodrigues, A David

    2017-05-15

    The role of bile salt export protein (BSEP) inhibition in drug-induced liver injury (DILI) has been investigated widely, while inhibition of the canalicular multidrug resistant protein 3 (MDR3) has received less attention. This transporter plays a pivotal role in secretion of phospholipids into bile and functions coordinately with BSEP to mediate the formation of bile acid-containing biliary micelles. Therefore, inhibition of MDR3 in human hepatocytes was examined across 125 drugs (70 of Most-DILI-concern and 55 of No-DILI-concern). Of these tested, 41% of Most-DILI-concern and 47% of No-DILI-concern drugs had MDR3 IC 50 values of <50 μM. A better distinction across DILI classifications occurred when systemic exposure was considered where safety margins of 50-fold had low sensitivity (0.29), but high specificity (0.96). Analysis of physical chemical property space showed that basic compounds were twice as likely to be MDR3 inhibitors as acids, neutrals, and zwitterions and that inhibitors were more likely to have polar surface area (PSA) values of <100 Å 2 and cPFLogD values between 1.5 and 5. These descriptors, with different cutoffs, also highlighted a group of compounds that shared dual potency as MDR3 and BSEP inhibitors. Nine drugs classified as Most-DILI-concern compounds (four withdrawn, four boxed warning, and one liver injury warning in their approved label) had intrinsic potency features of <20 μM in both assays, thereby reinforcing the notion that multiple inhibitory mechanisms governing bile formation (bile acid and phospholipid efflux) may confer additional risk factors that play into more severe forms of DILI as shown by others for BSEP inhibitors combined with multidrug resistance-associated protein (MRP2, MRP3, MRP4) inhibitory properties. Avoiding physical property descriptors that highlight dual BSEP and MDR3 inhibition or testing drug candidates for inhibition of multiple efflux transporters (e.g., BSEP, MDR3, and MRPs) may be an effective

  6. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

  7. Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

    Directory of Open Access Journals (Sweden)

    Ryan J Mailloux

    2010-10-01

    Full Text Available Uncoupling protein-2 (UCP2 is known to suppress mitochondrial reactive oxygen species (ROS production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

  8. Hili Inhibits HIV Replication in Activated T Cells.

    Science.gov (United States)

    Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

    2017-06-01

    P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements. Copyright © 2017 American Society for Microbiology.

  9. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-06-14

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

  10. Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

    International Nuclear Information System (INIS)

    Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.

    1988-01-01

    Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. [3H]leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis

  11. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  12. A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

    Science.gov (United States)

    DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

    2014-07-15

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. © 2014 DeMille et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. Kinetics of radiation-induced apoptosis in neonatal urogenital tissues with and without protein synthesis inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Gobe, G.C.; Harmon, B.; Schoch, E.; Allan, D.J. [Queensland Univ., St. Lucia, QLD (Australia). Dept. of Chemistry

    1996-12-31

    The difference in incidence of radiation-induced apoptosis between two neonatal urogenital tissues, kidney and testis, was analysed over a 24h period. Concurrent administration of cycloheximide (10mg/kg body weight), a protein synthesis inhibitor, with radiation treatment was used to determine whether new protein synthesis had a role in induction of apoptosis in this in vivo model. Many chemotherapeutic drugs act via protein synthesis inhibition, and we believe that the results of this latter analysis may provide information for the planning of concurrent radio and chemotherapy. Apoptosis was quantified using morphological parameters, and verified by DNA gel electrophoresis for the typical banding pattern, and by electron microscopy. The proliferative index in tissues was studied, using [6-{sup 3}H]-thymidine uptake ( 1h prior to euthanasia and collection of tissues) and autoradiography as indicators of cell proliferation (S-phase). Tissue was collected 2, 4, 6, 8, and 24h after radiation treatment. Expression of one of the apoptosis-associated genes, Bcl-2 (an apoptosis inhibitor/cell survival gene), was studied using immunohistochemistry. Apoptosis peaked at 4h in the testis and 6h in the kidney, emphasising the necessity of knowing tissue differences in radiation response if comparing changes at a particular time. A higher proportion (almost five fold) of the apoptotic cells died in S-phase in the kidney than the testis, over the 24h. Protein synthesis inhibition completely negated induction of apoptosis in both tissues. Necrosis was not identified at any time. Cycloheximide treatment greatly diminished Bcl-2 expression. The differences in response of the two tissues to irradiation relates to their innate cell (genetic) controls, which may be determined by their state of differentiation at time of treatment, or the tissue type. This in vivo study also suggests the model may be useful for analysis of other cancer therapies for example polychemotherapies or chemo

  14. Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

    DEFF Research Database (Denmark)

    Pratt-Riccio, Lilian Rose; Bianco, Cesare; Totino, Paulo Renato Rivas

    2011-01-01

    The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region h...

  15. A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

    Science.gov (United States)

    Drappier, Melissa; Elliott, Ruth; Zhang, Rong; Weiss, Susan R.; Silverman, Robert H.

    2018-01-01

    The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A. PMID:29652922

  16. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    International Nuclear Information System (INIS)

    Beall, G.N.; Chopra, I.J.; Solomon, D.H.; Kruger, S.R.

    1978-01-01

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  17. Progranulin, a major secreted protein of mouse adipose-derived stem cells, inhibits light-induced retinal degeneration.

    Science.gov (United States)

    Tsuruma, Kazuhiro; Yamauchi, Mika; Sugitani, Sou; Otsuka, Tomohiro; Ohno, Yuta; Nagahara, Yuki; Ikegame, Yuka; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

    2014-01-01

    Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

  18. Zinc finger protein 598 inhibits cell survival by promoting UV-induced apoptosis.

    Science.gov (United States)

    Yang, Qiaohong; Gupta, Romi

    2018-01-19

    UV is one of the major causes of DNA damage induced apoptosis. However, cancer cells adopt alternative mechanisms to evade UV-induced apoptosis. To identify factors that protect cancer cells from UV-induced apoptosis, we performed a genome wide short-hairpin RNA (shRNA) screen, which identified Zinc finger protein 598 (ZNF598) as a key regulator of UV-induced apoptosis. Here, we show that UV irradiation transcriptionally upregulates ZNF598 expression. Additionally, ZNF598 knockdown in cancer cells inhibited UV-induced apoptosis. In our study, we observe that ELK1 mRNA level as well as phosphorylated ELK1 levels was up regulated upon UV irradiation, which was necessary for UV irradiation induced upregulation of ZNF598. Cells expressing ELK1 shRNA were also resistant to UV-induced apoptosis, and phenocopy ZNF598 knockdown. Upon further investigation, we found that ZNF598 knockdown inhibits UV-induced apoptotic gene expression, which matches with decrease in percentage of annexin V positive cell. Similarly, ectopic expression of ZNF598 promoted apoptotic gene expression and also increased annexin V positive cells. Collectively, these results demonstrate that ZNF598 is a UV irradiation regulated gene and its loss results in resistance to UV-induced apoptosis.

  19. Sources of dietary protein in relation to blood pressure in a general Dutch population.

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    Wieke Altorf-van der Kuil

    Full Text Available BACKGROUND: Little is known about the relation of different dietary protein types with blood pressure (BP. We examined whether intake of total, plant, animal, dairy, meat, and grain protein was related to BP in a cross sectional cohort of 20,820 Dutch adults, aged 20-65 y and not using antihypertensive medication. DESIGN: Mean BP levels were calculated in quintiles of energy-adjusted protein with adjustment for age, sex, BMI, education, smoking, and intake of energy, alcohol, and other nutrients including protein from other sources. In addition, mean BP difference after substitution of 3 en% carbohydrates or MUFA with protein was calculated. RESULTS: Total protein and animal protein were not associated with BP (p(trend = 0.62 and 0.71 respectively, both at the expense of carbohydrates and MUFA. Systolic BP was 1.8 mmHg lower (p(trend36 g/d than in the lowest (<27 g/d quintile of plant protein. This inverse association was present both at the expense of carbohydrates and MUFA and more pronounced in individuals with untreated hypertension (-3.6 mmHg than in those with normal (+0.1 mmHg or prehypertensive BP (-0.3 mmHg; p(interaction<0.01. Meat and grain protein were not related to BP. Dairy protein was directly associated with systolic BP (+1.6 mmHg, p(trend<0.01, but not with diastolic BP (p(trend = 0.24. CONCLUSIONS: Total protein and animal protein were not associated with BP in this general untreated Dutch population. Plant protein may be beneficial to BP, especially in people with elevated BP. However, because high intake of plant protein may be a marker of a healthy diet and lifestyle in general, confirmation from randomized controlled trials is warranted.

  20. Identification of an Arabidopsis thaliana protein that binds to tomato mosaic virus genomic RNA and inhibits its multiplication

    International Nuclear Information System (INIS)

    Fujisaki, Koki; Ishikawa, Masayuki

    2008-01-01

    The genomic RNAs of positive-strand RNA viruses carry RNA elements that play positive, or in some cases, negative roles in virus multiplication by interacting with viral and cellular proteins. In this study, we purified Arabidopsis thaliana proteins that specifically bind to 5' or 3' terminal regions of tomato mosaic virus (ToMV) genomic RNA, which contain important regulatory elements for translation and RNA replication, and identified these proteins by mass spectrometry analyses. One of these host proteins, named BTR1, harbored three heterogeneous nuclear ribonucleoprotein K-homology RNA-binding domains and preferentially bound to RNA fragments that contained a sequence around the initiation codon of the 130K and 180K replication protein genes. The knockout and overexpression of BTR1 specifically enhanced and inhibited, respectively, ToMV multiplication in inoculated A. thaliana leaves, while such effect was hardly detectable in protoplasts. These results suggest that BTR1 negatively regulates the local spread of ToMV

  1. Inhibition of the SOS response of Escherichia coli by the Ada protein

    International Nuclear Information System (INIS)

    Vericat, J.A.; Guerrero, R.; Barbe, J.

    1988-01-01

    Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene

  2. Marine Longilenes, Oxasqualenoids with Ser-Thr Protein Phosphatase 2A Inhibition Activity

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    Francisco Cen-Pacheco

    2018-04-01

    Full Text Available The red seaweed Laurencia viridis is a rich source of oxygenated secondary metabolites that were derived from squalene. We report here the structures of three novel compounds, (+-longilene peroxide (1, longilene (2, and (+-prelongilene (3 that were isolated from this alga, in addition to other substances, 4 and 5, resulting from their acid-mediated degradation. The effect of compounds 1 and 3 against Ser-Thr protein phosphatase type 2A (PP2A was evaluated, showing that (+-longilene peroxide (1 inhibited PP2A (IC50 11.3 μM. In order to explain the interaction between PP2A and compounds 1 and 3, molecular docking simulations onto the PP2A enzyme-binding region were used.

  3. G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

    Science.gov (United States)

    Photowala, Huzefa; Blackmer, Trillium; Schwartz, Eric; Hamm, Heidi E; Alford, Simon

    2006-03-14

    Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

  4. Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

    Science.gov (United States)

    Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

    2007-07-01

    Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

  5. The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity

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    Dieter Wicher

    2007-12-01

    Full Text Available The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR, we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH: PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage.

  6. Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

    Science.gov (United States)

    Masselli, E; Carubbi, C; Gobbi, G; Mirandola, P; Galli, D; Martini, S; Bonomini, S; Crugnola, M; Craviotto, L; Aversa, F; Vitale, M

    2015-11-01

    Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

  7. Inhibition of transcription factor NF-kappaB signaling proteins IKKbeta and p65 through specific cysteine residues by epoxyquinone A monomer: correlation with its anti-cancer cell growth activity.

    Science.gov (United States)

    Liang, Mei-Chih; Bardhan, Sujata; Pace, Emily A; Rosman, Diana; Beutler, John A; Porco, John A; Gilmore, Thomas D

    2006-02-28

    Transcription factor NF-kappaB is constitutively active in many human chronic inflammatory diseases and cancers. Epoxyquinone A monomer (EqM), a synthetic derivative of the natural product epoxyquinol A, has previously been shown to be a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha)-induced activation of NF-kappaB, but the mechanism by which EqM inhibits NF-kappaB activation was not known. In this report, we show that EqM blocks activation of NF-kappaB by inhibiting two molecular targets: IkappaB kinase IKKbeta and NF-kappaB subunit p65. EqM inhibits TNF-alpha-induced IkappaBalpha phosphorylation and degradation by targeting IKKbeta, and an alanine substitution for Cys179 in the activation loop of IKKbeta makes it resistant to EqM-mediated inhibition. EqM also directly inhibits DNA binding by p65, but not p50; moreover, replacement of Cys38 in p65 with Ser abolishes EqM-mediated inhibition of DNA binding. Pretreatment of cells with reducing agent dithiothreitol dose-dependently reduces EqM-mediated inhibition of NF-kappaB, further suggesting that EqM directly modifies the thiol group of Cys residues in protein targets. Modifications of the exocyclic alkene of EqM substantially reduce EqM's ability to inhibit NF-kappaB activation. In the human SUDHL-4 lymphoma cell line, EqM inhibits both proliferation and NF-kappaB DNA binding, and activates caspase-3 activity. EqM also effectively inhibits the growth of human leukemia, kidney, and colon cancer cell lines in the NCI's tumor cell panel. Among six colon cancer cell lines, those with low amounts of constitutive NF-kappaB DNA-binding activity are generally more sensitive to growth inhibition by EqM. Taken together, these results suggest that EqM inhibits growth and induces cell death in tumor cells through a mechanism that involves inhibition of NF-kappaB activity at multiple steps in the signaling pathway.

  8. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  9. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

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    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  10. Exact parallel maximum clique algorithm for general and protein graphs.

    Science.gov (United States)

    Depolli, Matjaž; Konc, Janez; Rozman, Kati; Trobec, Roman; Janežič, Dušanka

    2013-09-23

    A new exact parallel maximum clique algorithm MaxCliquePara, which finds the maximum clique (the fully connected subgraph) in undirected general and protein graphs, is presented. First, a new branch and bound algorithm for finding a maximum clique on a single computer core, which builds on ideas presented in two published state of the art sequential algorithms is implemented. The new sequential MaxCliqueSeq algorithm is faster than the reference algorithms on both DIMACS benchmark graphs as well as on protein-derived product graphs used for protein structural comparisons. Next, the MaxCliqueSeq algorithm is parallelized by splitting the branch-and-bound search tree to multiple cores, resulting in MaxCliquePara algorithm. The ability to exploit all cores efficiently makes the new parallel MaxCliquePara algorithm markedly superior to other tested algorithms. On a 12-core computer, the parallelization provides up to 2 orders of magnitude faster execution on the large DIMACS benchmark graphs and up to an order of magnitude faster execution on protein product graphs. The algorithms are freely accessible on http://commsys.ijs.si/~matjaz/maxclique.

  11. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  12. In vivo inhibition of incorporation of (U-/sup 14/C)glucose into proteins in experimental focal epilepsy

    Energy Technology Data Exchange (ETDEWEB)

    Coutinho-Netto, J.; Boyar, M.M.; Abdul-Ghani, A.S.; Bradford, H.F.

    1982-08-01

    The in vivo incorporation of (/sup 14/C) from (U-/sup 14/C)-glucose into rat brain proteins from different cortical areas was examined in three different experimental focal epilepsies: cobalt, freeze-lesions, and tityustoxin. When (U-/sup 14/C)-glucose was injected intraperitoneally into awake and unrestrained animals with marked signs of epileptic hyperactivity, the inhibition of incorporation of (/sup 14/C)-amino acids into trichloracetic acid (TCA)-insoluble proteins was highest in the focal (sensorimotor) area when compared with distant regions (approx. 60%), but less when compared with the contralateral (sensorimotor) region (approx. 23%). Greatly decreased incorporation caused by both cobalt and freeze-lesion-induced epilepsies was also observed in the contralateral area when a comparison was made with distant regions (approx. 50%), but there were no significant differences in protein-specific radioactivity between the different distant areas.

  13. Revisiting the mechanistic basis of the French Paradox: red wine inhibits the activity of protein disulfide isomerase in vitro

    Science.gov (United States)

    Galinski, Christine N.; Zwicker, Jeffrey I.; Kennedy, Daniel R.

    2015-01-01

    Introduction Although epidemiologic evidence points to cardioprotective activity of red wine, the mechanistic basis for antithrombotic activity has not been established. Quercetin and related flavonoids are present in high concentrations in red but not white wine. Quercetin-glycosides were recently shown to prevent thrombosis in animal models through the inhibition of extracellular protein disulfide isomerase (PDI). We evaluated whether red or white wine inhibited PDI activity in vitro. Methods Quercetin levels in red and white wines were measured by HPLC analysis. Inhibition of PDI activity by red and white wines was assessed by an insulin reduction turbidity assay at various concentrations of wine. PDI inhibition was confirmed using a reduced peptide that contained a disulfide containing peptide as a substrate. The inhibition of PDI related thiol isomerases ERp5 and ERp57 was also assessed. Results We observed a dose-dependent decrease of PDI activity for a variety of red but not white wines. Red wine diluted to 3% final concentration resulted in over 80% inhibition of PDI activity by insulin reductase assay for all varieties tested. This inhibition was also observed in the peptide based assay. Red grape juice yielded similar results but ethanol alone did not affect PDI activity. Interestingly, red wine also inhibited the PDI related thiol isomerases ERp5 and ERp57, albeit to a lesser degree than PDI. Conclusions PDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption. PMID:26585763

  14. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-01-01

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. DOI: http://dx.doi.org/10.7554/eLife.24998.001 PMID:28613156

  15. Locally limited inhibition of bone resorption and orthodontic relapse by recombinant osteoprotegerin protein.

    Science.gov (United States)

    Schneider, D A; Smith, S M; Campbell, C; Hayami, T; Kapila, S; Hatch, N E

    2015-04-01

    To determine minimal dose levels required for local inhibition of orthodontic relapse by recombinant OPG protein (OPG-Fc), while also determining effects of injected OPG-Fc on alveolar bone and long bone. The Department of Orthodontics and Pediatric Dentistry at the University of Michigan. Eighteen male Sprague Dawley rats. Maxillary molars were moved with nickel-titanium springs and then allowed to relapse in Sprague Dawley rats. Upon appliance removal, animals were injected with a single dose of 1.0 mg/kg OPG-Fc, 0.1 mg/kg OPG-Fc, or phosphate-buffered saline (vehicle) just distal to the molar teeth. Tooth movement measurements were made from stone casts, which were scanned and digitally measured. Alveolar tissues were examined by histology. Micro-computed tomography was used to quantify changes in alveolar and femur bone. Local injection of OPG-Fc inhibited molar but not incisor relapse, when compared to vehicle-injected animals. No significant differences in alveolar or femur bone were seen between the three treatment groups after 24 days of relapse. Our results demonstrate that a single local injection of OPG-Fc effectively inhibits orthodontic relapse, with minimal systemic bone metabolic effects. Our results also show that a single injection of OPG-Fc will influence tooth movement only in teeth close to the injection site. These findings indicate that OPG-Fc has potential as a safe and effective pharmacological means to locally control osteoclasts, for uses such as maintaining anchorage during orthodontic tooth movement and preventing orthodontic relapse in humans. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

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    Marker Daniel F

    2012-11-01

    Full Text Available Abstract Background Human Immunodeficiency Virus-1 (HIV-1 associated neurocognitive disorders (HANDs are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART. While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2 as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i. We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence

  17. The lectin-like protein 1 in Lactobacillus rhamnosus GR-1 mediates tissue-specific adherence to vaginal epithelium and inhibits urogenital pathogens

    Science.gov (United States)

    Petrova, Mariya I.; Lievens, Elke; Verhoeven, Tine L. A.; Macklaim, Jean M.; Gloor, Gregory; Schols, Dominique; Vanderleyden, Jos; Reid, Gregor; Lebeer, Sarah

    2016-01-01

    The probiotic Lactobacillus rhamnosus GR-1 has been documented to survive implantation onto the vaginal epithelium and interfere with urogenital pathogens. However, the molecular mechanisms involved are largely unknown. Here, we report for the first time the construction of dedicated knock-out mutants in L. rhamnosus GR-1 to enable the study of gene functions. In a search for genes responsible for the adherence capacity of L. rhamnosus GR-1, a genomic region encoding a protein with homology to lectin-like proteins was identified. Phenotypic analyses of the knock-out mutant of L. rhamnosus GR-1 revealed a two-fold decreased adhesion to the vaginal and ectocervical epithelial cell lines compared to wild-type. In contrast, the adhesion to gastro-intestinal epithelial (Caco2) and endocervical cell lines (Hela and End1/E6E7) was not drastically affected by the mutation, suggesting that the LGR-1_Llp1 lectins mediates tissue tropism. The purified LGR-1_Llp1 protein also inhibited biofilm formation and adhesion of uropathogenic Escherichia coli. For the first time, an important role for a novel lectin-like protein in the adhesion capacity and host cell-specific interaction of a vaginal probiotic Lactobacillus strain has been discovered, with an additional role in pathogen inhibition. PMID:27869151

  18. The multi-targeted kinase inhibitor sorafenib inhibits enterovirus 71 replication by regulating IRES-dependent translation of viral proteins.

    Science.gov (United States)

    Gao, Meng; Duan, Hao; Liu, Jing; Zhang, Hao; Wang, Xin; Zhu, Meng; Guo, Jitao; Zhao, Zhenlong; Meng, Lirong; Peng, Yihong

    2014-06-01

    The activation of ERK and p38 signal cascade in host cells has been demonstrated to be essential for picornavirus enterovirus 71 (EV71) replication and up-regulation of virus-induced cyclooxygenase-2 (COX-2)/prostaglandins E2 (PGE2) expression. The aim of this study was to examine the effects of sorafenib, a clinically approved anti-cancer multi-targeted kinase inhibitor, on the propagation and pathogenesis of EV71, with a view to its possible mechanism and potential use in the design of therapy regimes for Hand foot and mouth disease (HFMD) patients with life threatening neurological complications. In this study, non-toxic concentrations of sorafenib were shown to inhibit the yield of infectious progeny EV71 (clinical BC08 strain) by about 90% in three different cell types. A similar inhibitory effect of sorafenib was observed on the synthesis of both viral genomic RNA and the VP1 protein. Interestingly, sorafenib exerted obvious inhibition of the EV71 internal ribosomal entry site (IRES)-mediated translation, the first step in picornavirus replication, by linking it to a firefly luciferase reporter gene. Sorafenib was also able to prevent both EV71-induced CPE and the activation of ERK and p38, which contributes to up-regulation COX-2/PGE2 expression induced by the virus. Overall, this study shows that sorafenib strongly inhibits EV71 replication at least in part by regulating viral IRES-dependent translation of viral proteins, indicating a novel potential strategy for the treatment of HFMD patients with severe neurological complications. To our knowledge, this is the first report that investigates the mechanism by which sorafenib inhibits EV71 replication. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  20. Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B.

    Science.gov (United States)

    Zhou, Jing; Chen, Jing; Zhang, Xiao-Min; Gao, Zhi-Can; Liu, Chun-Chun; Zhang, Yun-Na; Hou, Jin-Xiu; Li, Zhao-Yao; Kan, Lin; Li, Wen-Liang; Zhou, Bin

    2018-04-01

    Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S -transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B. IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo , but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities. Copyright © 2018 American Society for Microbiology.

  1. Suppression of Retinal Neovascularization in vivo by Inhibition of Vascular Endothelial Growth Factor (VEGF) Using Soluble VEGF-Receptor Chimeric Proteins

    Science.gov (United States)

    Aiello, Lloyd Paul; Pierce, Eric A.; Foley, Eliot D.; Takagi, Hitoshi; Chen, Helen; Riddle, Lavon; Ferrara, Napoleone; King, George L.; Smith, Lois E. H.

    1995-11-01

    The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-um section averaged 47% ± 4% (P < 0.001) and 37% ± 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66

  2. Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone

    International Nuclear Information System (INIS)

    Johnson, Timothy E.; Zhang, Xiaohua; Bleicher, Kimberly B.; Dysart, Gary; Loughlin, Amy F.; Schaefer, William H.; Umbenhauer, Diane R.

    2004-01-01

    Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential

  3. Lycopene Inhibits Metastasis of Human Liver Adenocarcinoma SK-Hep-1 Cells by Downregulation of NADPH Oxidase 4 Protein Expression.

    Science.gov (United States)

    Jhou, Bo-Yi; Song, Tuzz-Ying; Lee, Inn; Hu, Miao-Lin; Yang, Nae-Cherng

    2017-08-16

    NADPH oxidase 4 (NOX4), with the sole function to produce reactive oxygen species (ROS), can be a molecular target for disrupting cancer metastasis. Several studies have indicated that lycopene exhibited anti-metastatic actions in vitro and in vivo. However, the role of NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first confirmed the anti-metastatic effect of lycopene (0.1-5 μM) on human liver adenocarcinoma SK-Hep-1 cells. We showed that lycopene significantly inhibited NOX4 protein expression, with the strongest inhibition of 64.3 ± 10.2% (P lycopene. Lycopene also significantly inhibited NOX4 mRNA expression, NOX activity, and intracellular ROS levels in SK-Hep-1 cells. We then determined the effects of lycopene on transforming growth factor β (TGF-β)-induced metastasis. We found that TGF-β (5 ng/mL) significantly increased migration, invasion, and adhesion activity, the intracellular ROS level, matrix metalloproteinase 9 (MMP-9) and MMP-2 activities, the level of NOX4 protein expression, and NOX activity. All these TGF-β-induced effects were antagonized by the incubation of SK-Hep-1 cells with lycopene (2.5 μM). Using transient transfection of siRNA against NOX4, we found that the downregulation of NOX4 could mimic lycopene by inhibiting cell migration and the activities of MMP-9 and MMP-2 during the incubation with or without TGF-β on SK-Hep-1 cells. The results demonstrate that the downregulation of NOX4 plays a crucial role in the anti-metastatic action of lycopene in SK-Hep-1 cells.

  4. Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

    Science.gov (United States)

    Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

    2002-01-01

    The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431

  5. Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles.

    Science.gov (United States)

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

    2015-01-01

    The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A(-/-) mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.

  6. A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition

    Science.gov (United States)

    Rodriguez, Karl A.; Osmulski, Pawel A.; Pierce, Anson; Weintraub, Susan T.; Gaczynska, Maria; Buffenstein, Rochelle

    2015-01-01

    The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31y) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although HSP72 and HSP40 (Hdj1) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging. PMID:25018089

  7. Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

    Science.gov (United States)

    Chen, B; Han, B H; Sun, X H; Lim, R W

    1997-01-15

    We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

  8. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

    International Nuclear Information System (INIS)

    Moreau, P.L.

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

  9. TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1

    International Nuclear Information System (INIS)

    Wang Yuequn; Li Yongqing; Qi Xinzhu; Yuan Wuzhou; Ai Jianping; Zhu Chuanbing; Cao Lei; Yang Hong; Liu Fang; Wu Xiushan; Liu Mingyao

    2004-01-01

    The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted α-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway

  10. Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling

    DEFF Research Database (Denmark)

    Kermani, Abbas Jafari; Siersbaek, Majken S; Chen, Li

    2015-01-01

    for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo......Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments...

  11. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    International Nuclear Information System (INIS)

    Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

    2012-01-01

    Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  12. Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency

    DEFF Research Database (Denmark)

    Olpin, S E; Clark, S; Andresen, B S

    2005-01-01

    General mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl...... major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed....

  13. Prostacyclin Inhibits Non-Small Cell Lung Cancer Growth by a Frizzled 9-Dependent Pathway That Is Blocked by Secreted Frizzled-Related Protein 1

    Directory of Open Access Journals (Sweden)

    Meredith A. Tennis

    2010-03-01

    Full Text Available The goal of this study was to assess the ability of iloprost, an orally active prostacyclin analog, to inhibit transformed growth of human non-small cell lung cancer (NSCLC and to define the mechanism of iloprost's tumor suppressive effects. In a panel of NSCLC cell lines, the ability of iloprost to inhibit transformed cell growth was not correlated with the expression of the cell surface receptor for prostacyclin, but instead was correlated with the presence of Frizzled 9 (Fzd 9 and the activation of peroxisome proliferator-activated receptor-γ (PPARγ. Silencing of Fzd 9 blocked PPARγ activation by iloprost, and expression of Fzd 9 in cells lacking the protein resulted in iloprost's activation of PPARγ and inhibition of transformed growth. Interestingly, soluble Frizzled-related protein-1, a well-known inhibitor of Wnt/Fzd signaling, also blocked the effects of iloprost and Fzd 9. Moreover, mice treated with iloprost had reduced lung tumors and increased Fzd 9 expression. These studies define a novel paradigm, linking the eicosanoid pathway and Wnt signaling. In addition, these data also suggest that prostacyclin analogs may represent a new class of therapeutic agents in the treatment of NSCLC where the restoration of noncanonical Wnt signaling maybe important for the inhibition of transformed cell growth.

  14. Brazilin inhibits amyloid β-protein fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity

    Science.gov (United States)

    Du, Wen-Jie; Guo, Jing-Jing; Gao, Ming-Tao; Hu, Sheng-Quan; Dong, Xiao-Yan; Han, Yi-Fan; Liu, Fu-Feng; Jiang, Shaoyi; Sun, Yan

    2015-01-01

    Soluble amyloid β-protein (Aβ) oligomers, the main neurotoxic species, are predominantly formed from monomers through a fibril-catalyzed secondary nucleation. Herein, we virtually screened an in-house library of natural compounds and discovered brazilin as a dual functional compound in both Aβ42 fibrillogenesis inhibition and mature fibril remodeling, leading to significant reduction in Aβ42 cytotoxicity. The potent inhibitory effect of brazilin was proven by an IC50 of 1.5 +/- 0.3 μM, which was smaller than that of (-)-epigallocatechin gallate in Phase III clinical trials and about one order of magnitude smaller than those of curcumin and resveratrol. Most importantly, it was found that brazilin redirected Aβ42 monomers and its mature fibrils into unstructured Aβ aggregates with some β-sheet structures, which could prevent both the primary nucleation and the fibril-catalyzed secondary nucleation. Molecular simulations demonstrated that brazilin inhibited Aβ42 fibrillogenesis by directly binding to Aβ42 species via hydrophobic interactions and hydrogen bonding and remodeled mature fibrils by disrupting the intermolecular salt bridge Asp23-Lys28 via hydrogen bonding. Both experimental and computational studies revealed a different working mechanism of brazilin from that of known inhibitors. These findings indicate that brazilin is of great potential as a neuroprotective and therapeutic agent for Alzheimer's disease.

  15. Novel cyclo-peptides inhibit Ebola pseudotyped virus entry by targeting primed GP protein.

    Science.gov (United States)

    Li, Quanjie; Ma, Ling; Yi, Dongrong; Wang, Han; Wang, Jing; Zhang, Yongxin; Guo, Ying; Li, Xiaoyu; Zhou, Jinming; Shi, Yi; Gao, George F; Cen, Shan

    2018-07-01

    Ebola virus (EBOV) causes fatal hemorrhagic fever with high death rates in human. Currently, there are no available clinically-approved prophylactic or therapeutic treatments. The recently solved crystal structure of cleavage-primed EBOV glycoprotein (GPcl) in complex with the C domain of endosomal protein Niemann-Pick C1 (NPC1) provides a new target for the development of EBOV entry inhibitors. In this work, a computational approach using docking and molecular dynamic simulations is carried out for the rational design of peptide inhibitors. A novel cyclo-peptide (Pep-3.3) was identified to target at the late stage of EBOV entry and exhibit specific inhibitory activity against EBOV-GP pseudotyped viruses, with 50% inhibitory concentration (IC50) of 5.1 μM. In vitro binding assay and molecular simulations revealed that Pep-3.3 binds to GPcl with a KD value of 69.7 μM, through interacting with predicted residues in the hydrophobic binding pocket of GPcl. Mutation of predicted residues T83 caused resistance to Pep-3.3 inhibition in viral infectivity, providing preliminary support for the model of the peptide binding to GPcl. This study demonstrates the feasibility of inhibiting EBOV entry by targeting GPcl with peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. PRED-CLASS: cascading neural networks for generalized protein classification and genome-wide applications

    OpenAIRE

    Pasquier, Claude; Promponas, Vasilis; Hamodrakas, Stavros

    2009-01-01

    International audience; A cascading system of hierarchical, artificial neural networks (named PRED-CLASS) is presented for the generalized classification of proteins into four distinct classes-transmembrane, fibrous, globular, and mixed-from information solely encoded in their amino acid sequences. The architecture of the individual component networks is kept very simple, reducing the number of free parameters (network synaptic weights) for faster training, improved generalization, and the av...

  17. The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

    DEFF Research Database (Denmark)

    Porse, B T; Leviev, I; Mankin, A S

    1998-01-01

    A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine...... technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced...

  18. CASK inhibits ECV304 cell growth and interacts with Id1

    International Nuclear Information System (INIS)

    Qi Jie; Su Yongyue; Sun Rongju; Zhang Fang; Luo Xiaofeng; Yang Zongcheng; Luo Xiangdong

    2005-01-01

    Calcium/calmodulin-dependent serine protein kinase (CASK) is generally known as a scaffold protein. Here we show that overexpression of CASK resulted in a reduced rate of cell growth, while inhibition of expression of endogenous CASK via RNA-mediated interference resulted in an increased rate of cell growth in ECV304 cells. To explore the molecular mechanism, we identified a novel CASK-interacting protein, inhibitor of differentiation 1 (Id1) with a yeast two-hybrid screening. Furthermore, endogenous CASK and Id1 proteins were co-precipitated from the lysates of ECV304 cells by immunoprecipitation. Mammalian two-hybrid protein-protein interaction assays indicated that CASK possessed a different binding activity for Id1 and its alternative splicing variant. It is known that Id proteins play important roles in regulation of cell proliferation and differentiation. Thus, we speculate that the regulation of cell growth mediated by CASK may be involved in Id1. Our findings indicate a novel function of CASK, the mechanism that remains to be further investigated

  19. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    C Lin

    Full Text Available Our previous studies showed that bovine respiratory syncytial virus (BRSV followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2 epithelial cells with H. somni concentrated culture supernatant (CCS stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2 and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

  20. Alzheimer's associated β-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes.

    Directory of Open Access Journals (Sweden)

    Mitchell R White

    Full Text Available Accumulation of β-Amyloid (βA is a key pathogenetic factor in Alzheimer's disease; however, the normal function of βA is unknown. Recent studies have shown that βA can inhibit growth of bacteria and fungi. In this paper we show that βA also inhibits replication of seasonal and pandemic strains of H3N2 and H1N1 influenza A virus (IAV in vitro. The 42 amino acid fragment of βA (βA42 had greater activity than the 40 amino acid fragment. Direct incubation of the virus with βA42 was needed to achieve optimal inhibition. Using quantitative PCR assays βA42 was shown to reduce viral uptake by epithelial cells after 45 minutes and to reduce supernatant virus at 24 hours post infection. βA42 caused aggregation of IAV particles as detected by light transmission assays and electron and confocal microscopy. βA42 did not stimulate neutrophil H2O2 production or extracellular trap formation on its own, but it increased both responses stimulated by IAV. In addition, βA42 increased uptake of IAV by neutrophils. βA42 reduced viral protein synthesis in monocytes and reduced IAV-induced interleukin-6 production by these cells. Hence, we demonstrate for the first time that βA has antiviral activity and modulates viral interactions with phagocytes.

  1. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

    Science.gov (United States)

    Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

    2013-12-19

    The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression.

    Science.gov (United States)

    Liang, Xiaonan; Yong, Zheng; Su, Ruibin

    2018-06-11

    Opioid-induced respiratory depression is a major obstacle to improving the clinical management of moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  4. Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications.

    Science.gov (United States)

    Kobayashi, Hiroki; Harada, Hiroko; Nakamura, Masaomi; Futamura, Yushi; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-Ichiro; Shin-Ya, Kazuo; Doi, Takayuki; Takahashi, Takashi; Natsume, Tohru; Imoto, Masaya; Sakakibara, Yasubumi

    2012-04-05

    Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

  5. Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications

    Directory of Open Access Journals (Sweden)

    Kobayashi Hiroki

    2012-04-01

    Full Text Available Abstract Background Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. Results We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system. As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. Conclusions This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

  6. Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa.

    Science.gov (United States)

    Song, Yeong Hun; Uddin, Zia; Jin, Young Min; Li, Zuopeng; Curtis-Long, Marcus John; Kim, Kwang Dong; Cho, Jung Keun; Park, Ki Hun

    2017-12-01

    Protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase are important targets to treat obesity and diabetes, due to their deep correlation with insulin and leptin signalling, and glucose regulation. The methanol extract of Paulownia tomentosa fruits showed potent inhibition against both enzymes. Purification of this extract led to eight geranylated flavonoids (1-8) displaying dual inhibition of PTP1B and α-glucosidase. The isolated compounds were identified as flavanones (1-5) and dihydroflavonols (6-8). Inhibitory potencies of these compounds varied accordingly, but most of the compounds were highly effective against PTP1B (IC 50  = 1.9-8.2 μM) than α-glucosidase (IC 50  = 2.2-78.9 μM). Mimulone (1) was the most effective against PTP1B with IC 50  = 1.9 μM, whereas 6-geranyl-3,3',5,5',7-pentahydroxy-4'-methoxyflavane (8) displayed potent inhibition against α-glucosidase (IC 50  = 2.2 μM). All inhibitors showed mixed type Ι inhibition toward PTP1B, and were noncompetitive inhibitors of α-glucosidase. This mixed type behavior against PTP1B was fully demonstrated by showing a decrease in V max , an increase of K m , and K ik /K iv ratio ranging between 2.66 and 3.69.

  7. Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

    Science.gov (United States)

    Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

    2005-07-01

    We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

  8. AmyI-1-18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Fukuda, Shun; Sato, Teppei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-10-01

    In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4 × 10(-8) M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4 × 10(-6) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'-exonuclease activity

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Zanten, Gabriella van; Berenstein, Dvora

    2016-01-01

    We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development......-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated....

  10. Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

    Directory of Open Access Journals (Sweden)

    Caroline E. Moore

    2016-03-01

    Full Text Available Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1. A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.

  11. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    International Nuclear Information System (INIS)

    Zhou, Heng; Guo, Wei; Long, Cong; Wang, Huan; Wang, Jingchao; Sun, Xiaoping

    2015-01-01

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression

  12. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Heng [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Guo, Wei [Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Long, Cong; Wang, Huan; Wang, Jingchao [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Sun, Xiaoping, E-mail: xsun6@whu.edu.cn [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China)

    2015-01-16

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression.

  13. Inhibition of RNA Helicases of ssRNA+ Virus Belonging to Flaviviridae, Coronaviridae and Picornaviridae Families

    Directory of Open Access Journals (Sweden)

    Irene Briguglio

    2011-01-01

    Full Text Available Many viral pathogens encode the motor proteins named RNA helicases which display various functions in genome replication. General strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms: inhibition of the NTPase activity, by interferences with ATP binding and therefore by limiting the energy required for the unwinding and translocation, or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state; inhibition of nucleic acids binding to the helicase; inhibition of coupling of ATP hydrolysis to unwinding; inhibition of unwinding by sterically blocking helicase translocation. Recently, by in vitro screening studies, it has been reported that several benzotriazole, imidazole, imidazodiazepine, phenothiazine, quinoline, anthracycline, triphenylmethane, tropolone, pyrrole, acridone, small peptide, and Bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to Flaviviridae, Coronaviridae, and Picornaviridae families.

  14. Inhibition of breast cancer resistance protein (ABCG2 in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

    Directory of Open Access Journals (Sweden)

    Jun-O Jin

    Full Text Available Breast cancer resistance protein (ABCG2, a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs. ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.

  15. Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi *

    Science.gov (United States)

    Jia, Min; Li, Jianchao; Zhu, Jinwei; Wen, Wenyu; Zhang, Mingjie; Wang, Wenning

    2012-01-01

    GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gαi/o during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gαi·GDP. The crystal structures of Gαi·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gαi interaction features when compared with the only high resolution structure known with GL/Gαi interaction between RGS14 and Gαi1. Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gαi·GDP. A highly conserved “double Arg finger” sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gαi. Together with the sequence alignment, we suggest that the LGN GL/Gαi interaction represents a general binding mode between GL motifs and Gαi. We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors. PMID:22952234

  16. Involvement of MAPK proteins in bystander effects induced by chemicals and ionizing radiation

    International Nuclear Information System (INIS)

    Asur, Rajalakshmi; Balasubramaniam, Mamtha; Marples, Brian; Thomas, Robert A.; Tucker, James D.

    2010-01-01

    Many studies have examined bystander effects induced by ionizing radiation, however few have evaluated the ability of chemicals to induce similar effects. We previously reported the ability of two chemicals, mitomycin C (MMC) and phleomycin (PHL) to induce bystander effects in normal human lymphoblastoid cell lines. The focus of the current study was to determine the involvement of the MAPK proteins in bystander effects induced by physical and chemical DNA damaging agents and to evaluate the effects of MAPK inhibition on bystander-induced caspase 3/7 activation. The phosphorylation levels of the MAPK proteins ERK1/2, JNK, and p38, were measured from 1 to 24 h following direct or bystander exposure to MMC, PHL or radiation. We observed transient phosphorylation, at early time points, of all 3 proteins in bystander cells. We also evaluated the effect of MAPK inhibition on bystander-induced caspase 3/7 activity to determine the role of MAPK proteins in bystander-induced apoptosis. We observed bystander-induced activation of caspase 3/7 in bystander cells. Inhibition of MAPK proteins resulted in a decrease in caspase 3/7 activity at the early time points, and the caspase activity increased (in the case of ERK inhibition) or returned to basal levels (in the case of JNK or p38 inhibition) between 12 and 24 h. PHL is considered to be a radiomimetic agent, however in the present study PHL behaved more like a chemical and not like radiation in terms of MAPK phosphorylation. These results point to the involvement of MAPK proteins in the bystander effect induced by radiation and chemicals and provide additional evidence that this response is not limited to radiation but is a generalized stress response in cells.

  17. Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

    Science.gov (United States)

    Georges, Pauline; Boissart, Claire; Poulet, Aurélie; Peschanski, Marc; Benchoua, Alexandra

    2015-12-01

    Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro. © 2015 AlphaMed Press.

  18. Evidence for the involvement of a labile protein in stimulation of adrenal steroidogenesis under conditions not inhibitory to protein synthesis

    International Nuclear Information System (INIS)

    Krueger, R.J.; Orme-Johnson, N.R.

    1988-01-01

    Evidence is presented to support the hypothesis that synthesis of a labile protein is required for stimulation of steroidogenesis in rat adrenocortical cells. Amino acids L-canavanine and L-S-aminoethylcysteine, at concentrations as high as 5 mM, each inhibited steroidogenesis to a much greater extent than they inhibited protein synthesis. S-Aminoethylcysteine caused a 50% decrease in the stimulated rate of corticosterone production under conditions where incorporation of [35S]methionine into protein was unchanged. Both amino acids block stimulation of steroid synthesis at a step subsequent to the formation of cAMP and before the synthesis of progesterone. The onset of this effect, after the addition of the amino acids, on corticosterone production is quite rapid. These results provide support, that is not dependent on inhibition of protein synthesis, for the hypothesis that a labile protein mediates stimulation of steroidogenesis. Reversal of canavanine and S-aminoethylcysteine inhibition of steroidogenesis by arginine and lysine, respectively, suggests that the inhibitors are functioning as amino acid analogs. S-Aminoethylcysteine inhibits the incorporation of [3H]lysine into protein as well as inhibits steroidogenesis; further, [3H]S-aminoethylcysteine is incorporated into protein that is nonstimulatory. These results suggest that lysine residues play an essential role in the function of the labile protein or that the labile protein contains a large number of lysine residues

  19. 6-Hydroxydopamine Inhibits the Hepatitis C Virus through Alkylation of Host and Viral Proteins and the Induction of Oxidative Stress.

    Science.gov (United States)

    Lafreniere, Matthew A; Powdrill, Megan H; Singaravelu, Ragunath; Pezacki, John Paul

    2016-11-11

    Many viruses, including the hepatitis C virus (HCV), are dependent on the host RNA silencing pathway for replication. In this study, we screened small molecule probes, previously reported to disrupt loading of the RNA-induced silencing complex (RISC), including 6-hydroxydopamine (6-OHDA), suramin (SUR), and aurintricarboxylic acid (ATA), to examine their effects on viral replication. We found that 6-OHDA inhibited HCV replication; however, 6-OHDA was a less potent inhibitor of RISC than either SUR or ATA. By generating a novel chemical probe (6-OHDA-yne), we determined that 6-OHDA covalently modifies host and virus proteins. Moreover, 6-OHDA was shown to be an alkylating agent that is capable of generating adducts with a number of enzymes involved in the oxidative stress response. Furthermore, modification of viral enzymes with 6-OHDA and 6-OHDA-yne was found to inhibit their enzymatic activity. Our findings suggest that 6-OHDA is a probe for oxidative stress as well as protein alkylation, and these properties together contribute to the antiviral effects of this compound.

  20. Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions [v3; ref status: indexed, http://f1000r.es/50u

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2015-01-01

    Full Text Available Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52 derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html.

  1. D-Glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

    International Nuclear Information System (INIS)

    Oh, Hyun-Ji; Lee, Jason S.; Song, Dae-Kyu; Shin, Dong-Hoon; Jang, Byeong-Churl; Suh, Seong-Il; Park, Jong-Wook; Suh, Min-Ho; Baek, Won-Ki

    2007-01-01

    Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6 K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-Glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K

  2. Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

    International Nuclear Information System (INIS)

    Mattila, Elina; Marttila, Heidi; Sahlberg, Niko; Kohonen, Pekka; Tähtinen, Siri; Halonen, Pasi; Perälä, Merja; Ivaska, Johanna

    2010-01-01

    T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening

  3. The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

    Science.gov (United States)

    Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

    2001-07-01

    Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

  4. Exploiting the MDM2-CK1α Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    Science.gov (United States)

    Huart, Anne-Sophie; MacLaine, Nicola J.; Narayan, Vikram; Hupp, Ted R.

    2012-01-01

    Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between

  5. Exploiting the MDM2-CK1α protein-protein interface to develop novel biologics that induce UBL-kinase-modification and inhibit cell growth.

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Huart

    Full Text Available Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2 oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i ELISA with recombinant MDM2; (ii cell lysate pull-down towards endogenous MDM2; (iii MDM2-CK1α complex-based competition ELISA; and (iv MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii be used as a tool to study NEDDylation of CK1α, and (iii reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross

  6. Neogambogic acid prevents silica-induced fibrosis via inhibition of high-mobility group box 1 and MCP-1-induced protein 1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096 (China); Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Zhang, Mei, E-mail: meizhang1717@163.com [Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Wang, Zhongjiang [Department of Radiology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Cheng, Yusi; Liu, Haijun [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Zhou, Zewei [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Han, Bing [Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Chen, Baoan [Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Yao, Honghong, E-mail: yaohh@seu.edu.cn [Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096 (China); Chao, Jie, E-mail: chaojie@seu.edu.cn [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096 (China); Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China)

    2016-10-15

    Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO{sub 2}); early stages are characterized by alveolar inflammation, and later stages are characterized by progressive lung fibrosis. Mounting evidence indicates that high-mobility group box 1 (HMGB1) is involved in pulmonary fibrosis. Whether neogambogic acid (NGA) inhibits macrophage and fibroblast activation induced by SiO{sub 2} by targeting HMGB1 remains unclear. Methods and results: Experiments using cultured mouse macrophages (RAW264.7 cells) demonstrated that SiO{sub 2} treatment induces the expression of HMGB1 in a time- and dose-dependent manner via mitogen-activated protein kinases (MAPKs) and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway; in turn, this expression causes macrophage apoptosis and fibroblast activation. Pretreating macrophages with NGA inhibited the HMGB1 expression induced by SiO{sub 2} and attenuated both macrophage apoptosis and fibroblast activation. Moreover, NGA directly inhibited MCP-1-induced protein 1 (MCPIP1) expression, as well as markers of fibroblast activation and migration induced by SiO{sub 2}. Furthermore, the effects of NGA on macrophages and fibroblasts were confirmed in vivo by exposing mice to SiO{sub 2}. Conclusion: NGA can prevent SiO{sub 2}-induced macrophage activation and apoptosis via HMGB1 inhibition and SiO{sub 2}-induced fibrosis via the MCPIP1 pathway. Targeting HMGB1 and MCPIP1 with NGA could provide insights into the potential development of a therapeutic approach for alleviating the inflammation and fibrosis induced by SiO{sub 2}. - Highlights: • The SiO{sub 2} induced HMGB1 in alveolar macrophage and MCPIP1 in fibroblast. • NGA rescued the SiO{sub 2}-induced apoptosis of alveolar macrophages via HMGB1 signaling. • NGA inhibited the fibroblast activation induced by SiO{sub 2} via MCPIP1 signaling. • NGA might represent a potential therapeutic approach for silicosis.

  7. Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1α (Ero1α) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A*

    Science.gov (United States)

    Okumura, Masaki; Kadokura, Hiroshi; Hashimoto, Shoko; Yutani, Katsuhide; Kanemura, Shingo; Hikima, Takaaki; Hidaka, Yuji; Ito, Len; Shiba, Kohei; Masui, Shoji; Imai, Daiki; Imaoka, Susumu; Yamaguchi, Hiroshi; Inaba, Kenji

    2014-01-01

    Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. PMID:25122773

  8. Arborvitae (Thuja plicata essential oil significantly inhibited critical inflammation- and tissue remodeling-related proteins and genes in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Xuesheng Han

    2017-06-01

    Full Text Available Arborvitae (Thuja plicata essential oil (AEO is becoming increasingly popular in skincare, although its biological activity in human skin cells has not been investigated. Therefore, we sought to study AEO's effect on 17 important protein biomarkers that are closely related to inflammation and tissue remodeling by using a pre-inflamed human dermal fibroblast culture model. AEO significantly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1, intracellular cell adhesion molecule 1 (ICAM-1, interferon gamma-induced protein 10 (IP-10, interferon-inducible T-cell chemoattractant (I-TAC, monokine induced by interferon gamma (MIG, and macrophage colony-stimulating factor (M-CSF. It also showed significant antiproliferative activity and robustly inhibited collagen-I, collagen-III, plasminogen activator inhibitor-1 (PAI-1, and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2. The inhibitory effect of AEO on increased production of these protein biomarkers suggests it has anti-inflammatory property. We then studied the effect of AEO on the genome-wide expression of 21,224 genes in the same cell culture. AEO significantly and diversely modulated global gene expression. Ingenuity pathway analysis (IPA showed that AEO robustly affected numerous critical genes and signaling pathways closely involved in inflammatory and tissue remodeling processes. The findings of this study provide the first evidence of the biological activity and beneficial action of AEO in human skin cells.

  9. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  10. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  11. The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

    Directory of Open Access Journals (Sweden)

    Jasdave S Chahal

    Full Text Available Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

  12. Protein phosphatase 2A inhibition and circumvention of cisplatin cross-resistance by novel TCM-platinum anticancer agents containing demethylcantharidin.

    Science.gov (United States)

    To, Kenneth K W; Wang, Xinning; Yu, Chun Wing; Ho, Yee-Ping; Au-Yeung, Steve C F

    2004-09-01

    Novel TCM-platinum compounds [Pt(C(8)H(8)O(5))(NH(2)R)(2)] 1-5, derived from integrating demethylcantharidin, a modified component from a traditional Chinese medicine (TCM) with a platinum moiety, possess anticancer and protein phosphatase 2A inhibition properties. The compounds are able to circumvent cisplatin resistance by apparently targeting the DNA repair mechanism. Novel isosteric analogues [Pt(C(9)H(10)O(4))(NH(2)R)(2)] A and B, devoid of PP2A-inhibitory activity, were found to suffer from an enhanced DNA repair and were cross-resistant to cisplatin. The results advocate a well-defined structure-activity requirement associating the PP2A-inhibiting demethylcantharidin with the circumvention of cisplatin cross-resistance demonstrated by TCM-Pt compounds 1-5.

  13. Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

    Science.gov (United States)

    Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

    2017-09-01

    Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

    Science.gov (United States)

    Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan

    2015-03-25

    This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

  15. Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

    Science.gov (United States)

    Lambrecht, Jennifer A; Browne, Beth Ann; Downs, Diana M

    2010-11-05

    The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

  16. Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion

    DEFF Research Database (Denmark)

    Thams, P; Capito, K; Hedeskov, C J

    1990-01-01

    and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1...

  17. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes

    International Nuclear Information System (INIS)

    Hershko, A.; Rose, I.A.

    1987-01-01

    The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. The authors examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 125 I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: (i) Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown; (ii) release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent; (iii) direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown

  18. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

    Directory of Open Access Journals (Sweden)

    Li Q

    2013-07-01

    Full Text Available Qingli Li,1,2 Mark J Lambrechts,1 Qiuyang Zhang,1 Sen Liu,1 Dongxia Ge,1 Rutie Yin,2 Mingrong Xi,2 Zongbing You1 1Departments of Structural and Cellular Biology and Orthopaedic Surgery, Tulane Cancer Center and Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research and Regenerative Medicine, and Tulane Center for Aging, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, People’s Republic of China Abstract: Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA, are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy. Keywords: serine hydroxymethyltransferase, prostate cancer, apoptosis

  19. Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling▿ †

    Science.gov (United States)

    Stolp, Bettina; Abraham, Libin; Rudolph, Jochen M.; Fackler, Oliver T.

    2010-01-01

    Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2 (HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo. PMID:20147394

  20. Astrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription

    Directory of Open Access Journals (Sweden)

    Santoro Thomas J

    2005-02-01

    Full Text Available Abstract Background In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2 is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS. The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine. Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Methods Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin. Results The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti

  1. Astrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription.

    Science.gov (United States)

    Tomita, Michiyo; Holman, Brita J; Santoro, Christopher P; Santoro, Thomas J

    2005-02-25

    BACKGROUND: In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS). The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine.Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes. METHODS: Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin. RESULTS: The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti-oxidant, epigallocatechin

  2. CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction.

    Science.gov (United States)

    Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

    2013-12-15

    Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. © 2013.

  3. Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yun-Jeong; Cho, Hyun-Ji [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Magae, Junji [Magae Bioscience Institute, 49-4 Fujimidai, Tsukuba 300-1263 (Japan); Lee, In-Kyu [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Park, Keun-Gyu, E-mail: kpark@knu.ac.kr [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

    2013-12-15

    Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.

  4. PRED-CLASS: cascading neural networks for generalized protein classification and genome-wide applications.

    Science.gov (United States)

    Pasquier, C; Promponas, V J; Hamodrakas, S J

    2001-08-15

    A cascading system of hierarchical, artificial neural networks (named PRED-CLASS) is presented for the generalized classification of proteins into four distinct classes-transmembrane, fibrous, globular, and mixed-from information solely encoded in their amino acid sequences. The architecture of the individual component networks is kept very simple, reducing the number of free parameters (network synaptic weights) for faster training, improved generalization, and the avoidance of data overfitting. Capturing information from as few as 50 protein sequences spread among the four target classes (6 transmembrane, 10 fibrous, 13 globular, and 17 mixed), PRED-CLASS was able to obtain 371 correct predictions out of a set of 387 proteins (success rate approximately 96%) unambiguously assigned into one of the target classes. The application of PRED-CLASS to several test sets and complete proteomes of several organisms demonstrates that such a method could serve as a valuable tool in the annotation of genomic open reading frames with no functional assignment or as a preliminary step in fold recognition and ab initio structure prediction methods. Detailed results obtained for various data sets and completed genomes, along with a web sever running the PRED-CLASS algorithm, can be accessed over the World Wide Web at http://o2.biol.uoa.gr/PRED-CLASS.

  5. Overexpression of high molecular weight FGF-2 forms inhibits glioma growth by acting on cell-cycle progression and protein translation

    International Nuclear Information System (INIS)

    Lemiere, Sylvie; Azar, Rania; Belloc, Francis; Guersel, Demir; Pyronnet, Stephane; Bikfalvi, Andreas; Auguste, Patrick

    2008-01-01

    In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at the G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation

  6. Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

    Science.gov (United States)

    Okumura, Masaki; Kadokura, Hiroshi; Hashimoto, Shoko; Yutani, Katsuhide; Kanemura, Shingo; Hikima, Takaaki; Hidaka, Yuji; Ito, Len; Shiba, Kohei; Masui, Shoji; Imai, Daiki; Imaoka, Susumu; Yamaguchi, Hiroshi; Inaba, Kenji

    2014-09-26

    Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Lycopene depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase C in rat cerebrocortical nerve terminals.

    Science.gov (United States)

    Lu, Cheng-Wei; Hung, Chi-Feng; Jean, Wei-Horng; Lin, Tzu-Yu; Huang, Shu-Kuei; Wang, Su-Jane

    2018-05-01

    Lycopene is a natural dietary carotenoid that was reported to exhibit a neuroprotective profile. Considering that excitotoxicity and cell death induced by glutamate are involved in many brain disorders, the effect of lycopene on glutamate release in rat cerebrocortical nerve terminals and the possible mechanism involved in such effect was investigated. We observed here that lycopene inhibited 4-aminopyridine (4-AP)-evoked glutamate release and intrasynaptosomal Ca 2+ concentration elevation. The inhibitory effect of lycopene on 4-AP-evoked glutamate release was markedly reduced in the presence of the Ca v 2.2 (N-type) and Ca v 2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was insensitive to the intracellular Ca 2+ -release inhibitors dantrolene and CGP37157. Furthermore, in the presence of the protein kinase C inhibitors GF109203X and Go6976, the action of lycopene on evoked glutamate release was prevented. These results are the first to suggest that lycopene inhibits glutamate release from rat cortical synaptosomes by suppressing presynaptic Ca 2+ entry and protein kinase C activity.

  8. Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

    Science.gov (United States)

    Masci, Stefania; Laino, Paolo; Janni, Michela; Botticella, Ermelinda; Di Carli, Mariasole; Benvenuto, Eugenio; Danieli, Pier Paolo; Lilley, Kathryn S; Lafiandra, Domenico; D'Ovidio, Renato

    2015-04-22

    Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters.

  9. Design principles for cancer therapy guided by changes in complexity of protein-protein interaction networks.

    Science.gov (United States)

    Benzekry, Sebastian; Tuszynski, Jack A; Rietman, Edward A; Lakka Klement, Giannoula

    2015-05-28

    The ever-increasing expanse of online bioinformatics data is enabling new ways to, not only explore the visualization of these data, but also to apply novel mathematical methods to extract meaningful information for clinically relevant analysis of pathways and treatment decisions. One of the methods used for computing topological characteristics of a space at different spatial resolutions is persistent homology. This concept can also be applied to network theory, and more specifically to protein-protein interaction networks, where the number of rings in an individual cancer network represents a measure of complexity. We observed a linear correlation of R = -0.55 between persistent homology and 5-year survival of patients with a variety of cancers. This relationship was used to predict the proteins within a protein-protein interaction network with the most impact on cancer progression. By re-computing the persistent homology after computationally removing an individual node (protein) from the protein-protein interaction network, we were able to evaluate whether such an inhibition would lead to improvement in patient survival. The power of this approach lied in its ability to identify the effects of inhibition of multiple proteins and in the ability to expose whether the effect of a single inhibition may be amplified by inhibition of other proteins. More importantly, we illustrate specific examples of persistent homology calculations, which correctly predict the survival benefit observed effects in clinical trials using inhibitors of the identified molecular target. We propose that computational approaches such as persistent homology may be used in the future for selection of molecular therapies in clinic. The technique uses a mathematical algorithm to evaluate the node (protein) whose inhibition has the highest potential to reduce network complexity. The greater the drop in persistent homology, the greater reduction in network complexity, and thus a larger

  10. Comparative Proteomic Analysis Provides insight into the Key Proteins as Possible Targets Involved in Aspirin Inhibiting Biofilm Formation of Staphylococcus xylosus

    Directory of Open Access Journals (Sweden)

    Chang-Geng Xu

    2017-08-01

    Full Text Available Staphylococcus xylosus is an opportunistic pathogen that causes infection in humans and cow mastitis. And S. xylosus possesses a strong ability to form biofilms in vitro. As biofilm formation facilitates resistance to antimicrobial agents, the discovery of new medicinal properties for classic drugs is highly desired. Aspirin, which is the most common active component of non-steroidal anti-inflammatory compounds, affects the biofilm-forming capacity of various bacterial species. We have found that aspirin effectively inhibits biofilm formation of S. xylosus by Crystal violet (CV staining and scanning electron microscopy analyses. The present study sought to elucidate possible targets of aspirin in suppressing S. xylosus biofilm formation. Based on an isobaric tag for relative and absolute quantitation (iTRAQ fold-change of >1.2 or <0.8 (P-value < 0.05, 178 differentially expressed proteins, 111 down-regulated and 67 up-regulated, were identified after application of aspirin to cells at a 1/2 minimal inhibitory concentration. Gene ontology analysis indicated enrichment in metabolic processes for the majority of the differentially expressed proteins. We then used the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway database to analyze a large number of differentially expressed proteins and identified genes involved in biosynthesis of amino acids pathway, carbon metabolism (pentose phosphate and glycolytic pathways, tricarboxylic acid cycle and nitrogen metabolism (histidine metabolism. These novel proteins represent candidate targets in aspirin-mediated inhibition of S. xylosus biofilm formation at sub-MIC levels. The findings lay the foundation for further studies to identify potential aspirin targets.

  11. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

    Energy Technology Data Exchange (ETDEWEB)

    Benny Klimek, Margaret E.; Aydogdu, Tufan [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Link, Majik J.; Pons, Marianne [Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Koniaris, Leonidas G. [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology and Experimental Therapeutics Program, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL (United States); Zimmers, Teresa A., E-mail: tzimmers@med.miami.edu [Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology Program, Division of Surgical Oncology, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL (United States); Molecular Oncology and Experimental Therapeutics Program, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL (United States)

    2010-01-15

    Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

  12. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

    International Nuclear Information System (INIS)

    Benny Klimek, Margaret E.; Aydogdu, Tufan; Link, Majik J.; Pons, Marianne; Koniaris, Leonidas G.; Zimmers, Teresa A.

    2010-01-01

    Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

  13. How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations.

    Directory of Open Access Journals (Sweden)

    Tarja Äijänen

    2014-11-01

    Full Text Available Cholesteryl ester transfer protein (CETP mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL-cholesterol and decrease low density lipoprotein (LDL-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.

  14. Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

    Science.gov (United States)

    Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

    2015-01-01

    Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

  15. Physiological slowing and upregulation of inhibition in cortex are correlated with behavioral deficits in protein malnourished rats.

    Directory of Open Access Journals (Sweden)

    Rahul Chaudhary

    Full Text Available Protein malnutrition during early development has been correlated with cognitive and learning disabilities in children, but the neuronal deficits caused by long-term protein deficiency are not well understood. We exposed rats from gestation up to adulthood to a protein-deficient (PD diet, to emulate chronic protein malnutrition in humans. The offspring exhibited significantly impaired performance on the 'Gap-crossing' (GC task after reaching maturity, a behavior that has been shown to depend on normal functioning of the somatosensory cortex. The physiological state of the somatosensory cortex was examined to determine neuronal correlates of the deficits in behavior. Extracellular multi-unit recording from layer 4 (L4 neurons that receive direct thalamocortical inputs and layers 2/3 (L2/3 neurons that are dominated by intracortical connections in the whisker-barrel cortex of PD rats exhibited significantly low spontaneous activity and depressed responses to whisker stimulation. L4 neurons were more severely affected than L2/3 neurons. The response onset was significantly delayed in L4 cells. The peak response latency of L4 and L2/3 neurons was delayed significantly. In L2/3 and L4 of the barrel cortex there was a substantial increase in GAD65 (112% over controls and much smaller increase in NMDAR1 (12-20%, suggesting enhanced inhibition in the PD cortex. These results show that chronic protein deficiency negatively affects both thalamo-cortical and cortico-cortical transmission during somatosensory information processing. The findings support the interpretation that sustained protein deficiency interferes with features of cortical sensory processing that are likely to underlie the cognitive impairments reported in humans who have suffered from prolonged protein deficiency.

  16. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    Directory of Open Access Journals (Sweden)

    Li J

    2015-02-01

    Full Text Available Jie Li,1 Chao Zhang,1 Hongchuan Jiang,1 Jiao Cheng21Department of General Surgery, 2Department of Gynaecology and Obstetrics, Beijing Chao-Yang Hospital, Beijing, People’s Republic of ChinaAbstract: Hypoxia-inducible factor-1 (HIF-1 is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10-7 mol/L, by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future.Keywords: Andrographolide (Andro, HIF-1α, inhibit, breast cancer, hypoxia, PI3k/AKT/mTOR pathway

  17. Mapping the binding interface between an HIV-1 inhibiting intrabody and the viral protein Rev.

    Directory of Open Access Journals (Sweden)

    Thomas Vercruysse

    Full Text Available HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb190 as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb190-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb190 and the Rev multimerization domains were evaluated in different assays measuring Nb190-Rev interaction or viral production. Seven residues within Nb190 and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb190-Rev structural interface. This Nb190-Rev interaction model can guide further studies of the Nb190 effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb190 epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.

  18. Inhibition of the pore-forming protein perforin by a series of aryl-substituted isobenzofuran-1(3H)-ones.

    Science.gov (United States)

    Spicer, Julie A; Huttunen, Kristiina M; Miller, Christian K; Denny, William A; Ciccone, Annette; Browne, Kylie A; Trapani, Joseph A

    2012-02-01

    An aryl-substituted isobenzofuran-1(3H)-one lead compound was identified from a high throughput screen designed to find inhibitors of the lymphocyte pore-forming protein perforin. A series of analogs were then designed and prepared, exploring structure-activity relationships through variation of 2-thioxoimidazolidin-4-one and furan subunits on an isobenzofuranone core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 natural killer effector cells was determined. Several compounds showed excellent activity at concentrations that were non-toxic to the killer cells. This series represents a significant improvement on previous classes of compounds, being substantially more potent and largely retaining activity in the presence of serum. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-κB in H1299 human lung cancer cells

    International Nuclear Information System (INIS)

    Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

    2009-01-01

    Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (Gαi1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of Gαi1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of Gαi1QL. Gαi1 induced the transcription of Bcl-2 by activation of NF-κB, which resulted from an increase in NF-κB p50 protein. We conclude that Gαi1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-κB activation.

  20. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    Directory of Open Access Journals (Sweden)

    Kim CH

    2016-05-01

    Full Text Available Cy Hyun Kim,1,2,* Jin-Hong Shin,1,3,* Sung Jun Hwang,1,2 Yung Hyun Choi,4 Dae-Seong Kim,1,3 Cheol Min Kim2,51Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, 2Center for Anti-Aging Industry, Pusan National University, Busan, 3Department of Neurology, Pusan National University Yangsan Hospital, Yangsan, 4Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan, 5Department of Biomedical Informatics, Pusan National University School of Medicine, Yangsan, Republic of Korea*These authors contributed equally to this work Abstract: Schisandrae fructus (SF has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 µg/mL of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 µg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged

  1. Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

    Science.gov (United States)

    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

    2016-06-01

    Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

  2. Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase.

    Science.gov (United States)

    Gao, Yuanyuan; Fotovati, Abbas; Lee, Cathy; Wang, Michelle; Cote, Gilbert; Guns, Emma; Toyota, Brian; Faury, Damien; Jabado, Nada; Dunn, Sandra E

    2009-12-01

    Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where 5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes approximately 15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM.

  3. 5,7-Dimethoxycoumarin prevents chronic mild stress induced depression in rats through increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels

    Directory of Open Access Journals (Sweden)

    Wei Yang

    2018-02-01