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Sample records for binding protein creb

  1. Oct-1 potentiates CREB-driven cyclin D1 promoter activation via a phospho-CREB- and CREB binding protein-independent mechanism.

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    Boulon, Séverine; Dantonel, Jean-Christophe; Binet, Virginie; Vié, Annick; Blanchard, Jean-Marie; Hipskind, Robert A; Philips, Alexandre

    2002-11-01

    Cyclin D1, the regulatory subunit for mid-G(1) cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals.

  2. CREB binding protein is required for both short-term and long-term memory formation.

    NARCIS (Netherlands)

    Chen, G.; Zou, X.; Watanabe, H.; Deursen, J.M.A. van; Shen, J.

    2010-01-01

    CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated

  3. Roles of phosphorylation and helix propensity in the binding of the KIX domain of CREB-binding protein by constitutive (c-Myb) and inducible (CREB) activators.

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    Zor, Tsaffrir; Mayr, Bernhard M; Dyson, H Jane; Montminy, Marc R; Wright, Peter E

    2002-11-01

    cAMP-response element-binding protein (CREB)-binding protein (CBP) is a general transcriptional co-activator that mediates interactions between transcription factors and the basal transcription machinery. To obtain insights into the mechanism by which the KIX domain of CBP can recognize the transactivation domains of many different transcription factors, we have used NMR and biochemical analyses to study the interactions of KIX with the transactivation domain from the constitutive activator c-Myb and with the kinase-inducible transactivation domain (KID) from CREB. NMR chemical shift mapping shows that both activation domains bind to the same surface of KIX. In the unbound state, both the phosphorylated KID and c-Myb activation domains are only partly structured, and binding to KIX is coupled with folding to form an amphipathic helix. Helix-destabilizing mutations significantly impair binding, whereas mutations that increase the intrinsic secondary structure content of the free phosphorylated KID peptide have only a small influence on binding affinity. Low affinity but specific binding of unphosphorylated KID to KIX was measured by ITC and was also observed in Western blot assays and by a fluorescence resonance energy transfer experiment in living cells. The large increase in the affinity for phosphorylated KID is due to favorable intermolecular interactions involving the phosphate moiety. After induction by phosphorylation, CREB is able to compete effectively with other transcriptional activators for binding to CBP.

  4. Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB

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    Mayr, Bernhard M.; Canettieri, Gianluca; Montminy, Marc R.

    2001-01-01

    Ser-133 phosphorylation of the cAMP-responsive element-binding protein (CREB) is sufficient to induce cellular gene expression in response to cAMP, but additional promoter-bound factors are required for target gene activation by CREB in response to mitogen/stress signals. To compare the relative effects of different signals on recruitment of the coactivator CREB-binding protein (CBP) to CREB in living cells, we developed a fluorescence resonance energy transfer (FRET) assay. cAMP promoted the interaction of CREB with CBP in a phosphorylation-dependent manner by FRET analysis, but mitogen/stress signals were far less effective in stimulating complex formation even though they induced comparable levels of Ser-133 phosphorylation. cAMP and non-cAMP stimuli were comparably active in promoting this interaction in the cytosol; the formation of CREB⋅CBP complexes in response to non-cAMP signals was specifically inhibited in the nucleus. Non-cAMP signals had no effect on intrinsic CREB- or CBP-binding activities by Far Western blot assay, thereby supporting the presence of a distinct CREB⋅CBP antagonist. Our studies indicate that the relative effects of cAMP and mitogen/stress signals on CREB⋅CBP complex formation impart selectivity to gene activation through CREB phosphorylated at Ser-133. PMID:11535812

  5. Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.

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    Clem, Brian F; Hudson, Elizabeth A; Clark, Barbara J

    2005-03-01

    Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

  6. 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

  7. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

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    Kovács, Krisztián A; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J; Cardinaux, Jean-René

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  9. Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia

    NARCIS (Netherlands)

    van der Sligte, Naomi E.; Kampen, Kim R.; ter Elst, Arja; Scherpen, Frank J. G.; Meeuwsen-de Boer, Tiny G. J.; Guryev, Victor; van Leeuwen, Frank N.; Kornblau, Steven M.; de Bont, Eveline S. J. M.

    2015-01-01

    Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric

  10. "Nuclear FGF receptor-1 and CREB binding protein: an integrative signaling module".

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    Stachowiak, Michal K; Birkaya, B; Aletta, J M; Narla, S T; Benson, C A; Decker, B; Stachowiak, E K

    2015-05-01

    In this review we summarize the current understanding of a novel integrative function of Fibroblast Growth Factor Receptor-1 (FGFR1) and its partner CREB Binding Protein (CBP) acting as a nuclear regulatory complex. Nuclear FGFR1 and CBP interact with and regulate numerous genes on various chromosomes. FGFR1 dynamic oscillatory interactions with chromatin and with specific genes, underwrites gene regulation mediated by diverse developmental signals. Integrative Nuclear FGFR1 Signaling (INFS) effects the differentiation of stem cells and neural progenitor cells via the gene-controlling Feed-Forward-And-Gate mechanism. Nuclear accumulation of FGFR1 occurs in numerous cell types and disruption of INFS may play an important role in developmental disorders such as schizophrenia, and in metastatic diseases such as cancer. Enhancement of INFS may be used to coordinate the gene regulation needed to activate cell differentiation for regenerative purposes or to provide interruption of cancer stem cell proliferation. © 2014 Wiley Periodicals, Inc.

  11. Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

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    Ferah Yildirim

    Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

  12. Glutamine rich and basic region/leucine zipper (bZIP) domains stabilize cAMP-response element-binding protein (CREB) binding to chromatin.

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    Mayr, Bernhard M; Guzman, Ernesto; Montminy, Marc

    2005-04-15

    We have examined the dynamics of cAMP-response element-binding protein (CREB) binding to chromatin in live cells using fluorescence recovery after photobleaching (FRAP). CREB was found to bind to target sites with a residence time of 100 s, and exposure to a cAMP agonist had no effect on these kinetics. In addition to the basic region/leucine zipper (bZIP) domain, a glutamine-rich trans-activation domain in CREB called Q2 also appeared to be critical for promoter occupancy. Indeed, mutations in Q2 that reduced residence time by FRAP assay disrupted target gene activation via CREB in cells exposed to a cAMP agonist. Notably, insertion of the glutamine-rich B trans-activation domain of SP1 into a mutant CREB polypeptide lacking Q2 stabilized CREB occupancy and rescued target gene activation. These results suggest a novel mechanism by which the family of glutamine-rich activators promotes cellular gene expression.

  13. Multiple functions of CREB-binding protein during postembryonic development: identification of target genes.

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    Roy, Amit; George, Smitha; Palli, Subba Reddy

    2017-12-29

    Juvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect. CBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc. These data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.

  14. CREB-binding protein, p300, butyrate, and Wnt signaling in colorectal cancer.

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    Bordonaro, Michael; Lazarova, Darina L

    2015-07-21

    This paper reviews the distinctive roles played by the transcriptional coactivators CREB-binding protein (CBP) and p300 in Wnt/β-catenin signaling and cell physiology in colorectal cancer (CRC). Specifically, we focus on the effects of CBP- and p300-mediated Wnt activity on (1) neoplastic progression; (2) the activities of butyrate, a breakdown product of dietary fiber, on cell signaling and colonic cell physiology; (3) the development of resistance to histone deacetylase inhibitors (HDACis), including butyrate and synthetic HDACis, in colonic cells; and (4) the physiology and number of cancer stem cells. Mutations of the Wnt/β-catenin signaling pathway initiate the majority of CRC cases, and we have shown that hyperactivation of this pathway by butyrate and other HDACis promotes CRC cell apoptosis. This activity by butyrate may in part explain the preventive action of fiber against CRC. However, individuals with a high-fiber diet may still develop neoplasia; therefore, resistance to the chemopreventive action of butyrate likely contributes to CRC. CBP or p300 may modify the ability of butyrate to influence colonic cell physiology since the two transcriptional coactivators affect Wnt signaling, and likely, its hyperactivation by butyrate. Also, CBP and p300 likely affect colonic tumorigenesis, as well as stem cell pluripotency. Improvement of CRC prevention and therapy requires a better understanding of the alterations in Wnt signaling and gene expression that underlie neoplastic progression, stem cell fate, and the development of resistance to butyrate and clinically relevant HDACis. Detailed knowledge of how CBP- and p300 modulate colonic cell physiology may lead to new approaches for anti-CRC prevention and therapeutics, particularly with respect to combinatorial therapy of CBP/p300 inhibitors with HDACis.

  15. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

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    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  16. Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1 Expression by Orphan Nuclear Receptor Nr4a1

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    Michael P. Greenwood

    2017-12-01

    Full Text Available Cyclic AMP (cAMP inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1 is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH. We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

  17. Transgenic Mice Expressing a Truncated Form of CREB-Binding Protein (CBP) Exhibit Deficits in Hippocampal Synaptic Plasticity and Memory Storage

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    Wood, Marcelo A.; Kaplan, Michael P.; Park, Alice; Blanchard, Edward J.; Oliveira, Ana M. M.; Lombardi, Thomas L.; Abel, Ted

    2005-01-01

    Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII[alpha] promoter drives…

  18. The transcriptional cofactor MIER1-beta negatively regulates histone acetyltransferase activity of the CREB-binding protein

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    Gillespie Laura L

    2008-08-01

    Full Text Available Abstract Background Mier1 encodes a novel transcriptional regulator and was originally isolated as a fibroblast growth factor early response gene. Two major protein isoforms have been identified, MIER1α and β, which differ in their C-terminal sequence. Previously, we demonstrated that both isoforms recruit histone deacetylase 1 (HDAC1 to repress transcription. To further explore the role of MIER1 in chromatin remodeling, we investigated the functional interaction of MIER1 with the histone acetyltransferase (HAT, Creb-binding protein (CBP. Findings Using GST pull-down assays, we demonstrate that MIER1 interacts with CBP and that this interaction involves the N-terminal half (amino acids 1–283 of MIER1, which includes the acidic activation and ELM2 domains and the C-terminal half (amino acids 1094–2441 of CBP, which includes the bromo-, HAT, C/H3 and glutamine-rich domains. Functional analysis, using HEK293 cells, shows that the CBP bound to MIER1 in vivo has no detectable HAT activity. Histone 4 peptide binding assays demonstrate that this inhibition of HAT activity is not the result of interference with histone binding. Conclusion Our data indicate that an additional mechanism by which MIER1 could repress transcription involves the inhibition of histone acetyltransferase activity.

  19. CREB Binding Protein Interacts with Nucleoporin-Specific FG Repeats That Activate Transcription and Mediate NUP98-HOXA9 Oncogenicity

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    Kasper, Lawryn H.; Brindle, Paul K.; Schnabel, Catherine A.; Pritchard, Colin E. J.; Cleary, Michael L.; van Deursen, Jan M. A.

    1999-01-01

    Genes encoding the Phe-Gly (FG) repeat-containing nucleoporins NUP98 and CAN/NUP214 are at the breakpoints of several chromosomal translocations associated with human acute myeloid leukemia (AML), but their role in oncogenesis is unclear. Here we demonstrate that the NUP98-HOXA9 fusion gene encodes two nuclear oncoproteins with either 19 or 37 NUP98 FG repeats fused to the DNA binding and PBX heterodimerization domains of the transcription factor HOXA9. Both NUP98-HOXA9 chimeras transformed NIH 3T3 fibroblasts, and this transformation required the HOXA9 domains for DNA binding and PBX interaction. Surprisingly, the FG repeats acted as very potent transactivators of gene transcription. This NUP98-derived activity is essential for transformation and can be replaced by the bona fide transactivation domain of VP16. Interestingly, FG repeat-containing segments derived from the nucleoporins NUP153 and CAN/NUP214 functioned similarly to those from NUP98. We further demonstrate that transactivation by FG repeat-rich segments of NUP98 correlates with their ability to interact functionally and physically with the transcriptional coactivators CREB binding protein (CBP) and p300. This finding shows, for the first time, that a translocation-generated fusion protein appears to recruit CBP/p300 as an important step of its oncogenic mechanism. Together, our results suggest that NUP98-HOXA9 chimeras are aberrant transcription factors that deregulate HOX-responsive genes through the transcriptional activation properties of nucleoporin-specific FG repeats that recruit CBP/p300. Indeed, FG repeat-mediated transactivation may be a shared pathogenic function of nucleoporins implicated human AML. PMID:9858599

  20. Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

    International Nuclear Information System (INIS)

    Kwon, Yong-Jun; Sun, Yuanjie; Kim, Nam-Ho; Huh, Sung-Oh

    2009-01-01

    Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.

  1. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

    International Nuclear Information System (INIS)

    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M.; Heery, David M.

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBPΔ998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  2. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

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    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M. [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Heery, David M., E-mail: david.heery@nottingham.ac.uk [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  3. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

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    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter.

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    Rogge, George A; Shen, Li-Ling; Kuhar, Michael J

    2010-07-16

    Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  5. Structural Analysis of a Complex between Small Ubiquitin-like Modifier 1 (SUMO1) and the ZZ Domain of CREB-binding Protein (CBP/p300) Reveals a New Interaction Surface on SUMO

    DEFF Research Database (Denmark)

    Diehl, Carl; Akke, Mikael; Bekker-Jensen, Simon

    2016-01-01

    We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a ...

  6. Insulin-like growth factor-1 (IGF-1 induces the activation/phosphorylation of Akt kinase and cAMP response element-binding protein (CREB by activating different signaling pathways in PC12 cells

    Directory of Open Access Journals (Sweden)

    Zheng Wen-Hua

    2006-06-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is a polypeptide growth factor with a variety of functions in both neuronal and non-neuronal cells. IGF-1 plays anti-apoptotic and other functions by activating multiple signaling pathways including Akt kinase, a serine/threonine kinase essential for cell survival. The nuclear transcription factor cAMP response element-binding protein (CREB may also be involved although relationships between these two proteins in IGF-1 receptor signaling and protection is not clear, especially in neuronal cells. Results IGF-1, in a concentration- and time-dependent manner, induces the activation/phosphorylation of Akt and CREB in PC12 cells by activating different signaling pathways. IGF-1 induced a sustained phosphorylation of Akt while only a transient one was seen for CREB. The phosphorylation of Akt is mediated by the PI3 kinase pathway while that of CREB is dependent on the activation of both MAPK kinase and p38 MAPK. Moreover, the stimulation of PKC attenuated the phosphorylation of Akt induced by IGF-1 while enhancing that of CREB. Survival assays with various kinase inhibitors suggested that the activation/phosphorylation of both Akt and CREB contributes to IGF-1 mediated cell survival in PC12 cells. Conclusion These data suggest that IGF-1 induced the activation of Akt and CREB using distinct pathways in PC12 cells.

  7. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  8. Ultraviolet B (UVB) induction of the c-fos promoter is mediated by phospho-cAMP response element binding protein (CREB) binding to CRE and c-fos activator protein 1 site (FAP1) cis elements.

    Science.gov (United States)

    Gonzales, Melissa; Bowden, G Tim

    2002-06-26

    The ultraviolet B (UVB) portion (280-320 nm) of the ultraviolet spectrum has been shown to contribute to the development of non-melanoma skin cancer in humans. Research in the human keratinocyte cell line, HaCaT, revealed that UVB irradiation caused the upregulation of the transcription factor activator protein-1 (AP-1). The AP-1 complex formed in UVB-irradiated HaCaT cells is specifically composed of c-fos and Jun D. c-Fos expression was induced in a manner that correlated with the UVB-induced activation of AP-1. To investigate how c-fos expression is regulated by UVB irradiation, the role of each of four cis elements within the c-fos promoter was evaluated. Clustered point mutations at the sis inducible element (SIE), serum response element (SRE), c-fos AP-1 site (FAP1), or cyclic AMP response elements (CRE) significantly inhibited UVB induction of the c-fos promoter. This indicated that all four cis elements are required for maximum promoter activity. The CRE and FAP1 elements were the two most active cis elements that mediate the UVB transactivation of c-fos. Homodimers of phosphorylated cAMP response element binding protein (CREB) were induced by UVB irradiation to bind to each of these elements. Therefore, CREB may function as an important regulatory protein in the UVB-induced expression of c-fos.

  9. cAMP-response Element-binding Protein (CREB) and NF-κB Transcription Factors Are Activated during Prolonged Hypoxia and Cooperatively Regulate the Induction of Matrix Metalloproteinase MMP1*

    Science.gov (United States)

    Nakayama, Koh

    2013-01-01

    Responses to low levels of oxygen (hypoxia) are essential to maintain homeostasis. During the hypoxic response, gene expression is altered by various transcription factors. The transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxic response. The α subunit of HIF, which is actively degraded during normoxia, becomes stabilized during hypoxia, which leads to HIF activation. A microarray analysis of HeLa cells showed that expression of matrix metalloproteinase 1 (MMP1) was markedly induced during prolonged hypoxia. CREB and NF-κB binding sites were identified in the MMP1 promoter region between 1945 and 1896 nucleotides upstream of the transcription start site. Assays with luciferase reporters demonstrated that HIF activity was induced during the early phase of hypoxia, whereas CREB and NF-κB were activated during the later (prolonged) phase. Depletion of CREB and/or NF-κB reduced MMP1 induction during prolonged hypoxia both at the mRNA and protein levels. A chromatin immunoprecipitation assay demonstrated binding of CREB and NF-κB to the MMP1 promoter. Finally, cell migration and invasion on a collagen matrix and pulmonary metastasis in nude mice were inhibited after depletion of CREB and NF-κB in MDA-MB-231 cells. Taken together, these results suggest that the cooperative action of CREB and NF-κB plays an important role to induce MMP1 expression during prolonged hypoxia and regulates cell migration and invasion in cancer cells. PMID:23775082

  10. Cyclic AMP-Responsive Element-Binding Protein (CREB is Critical in Autoimmunity by Promoting Th17 but Inhibiting Treg Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Xiaohu Wang

    2017-11-01

    Full Text Available The molecular mechanisms that govern differential T cell development into pro-inflammatory Th17 vs. regulatory T (Treg cells remain unclear. Here, we show that selective deletion of CREB in T cells or Th17 cells impaired Th17 cell differentiation in vitro and in vivo, and led to resistance to autoimmune diseases. Mechanistically, CREB, activated by CD3-PKC-ϴ signaling, plays a key role in regulating Th17 cell differentiation, at least in part through directly binding to the Il17-Il17f gene locus. Unexpectedly, although dispensable for FOXP3 expression and for the homeostasis and suppressive function of thymus-derived Treg cells, CREB negatively regulates the survival of TGF-β-induced Treg cells, and deletion of CREB resulted in increased FOXP3+ Treg cells in the intestine and protection in a colitis model. Thus, CREB is critical in autoimmune diseases by promoting Th17 cell and inhibiting de novo Treg cell generation.

  11. The Study of Posttranslational Modifications of Tau Protein by Nuclear Magnetic Resonance Spectroscopy: Phosphorylation of Tau Protein by ERK2 Recombinant Kinase and Rat Brain Extract, and Acetylation by Recombinant Creb-Binding Protein.

    Science.gov (United States)

    Qi, Haoling; Despres, Clément; Prabakaran, Sudhakaran; Cantrelle, François-Xavier; Chambraud, Béatrice; Gunawardena, Jeremy; Lippens, Guy; Smet-Nocca, Caroline; Landrieu, Isabelle

    2017-01-01

    Nuclear magnetic resonance (NMR) spectroscopy can be used as an analytical tool to investigate posttranslational modifications of protein. NMR is a valuable tool to map the interaction regions of protein partners. Here, we present protocols that have been developed in the course of our studies of the neuronal Tau protein. Tau is found aggregated in the neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation and acetylation of Tau. We have used NMR to investigate how these posttranslational modifications of Tau affect the interactions with its partners. We present here detailed protocols of in vitro phosphorylation of Tau by recombinant kinase, ERK2, or kinase activity of rat brain extracts, and acetylation by recombinant Creb-binding protein (CBP) acetyltransferase. The analytical characterization of the modified Tau by NMR spectroscopy is additionally described.

  12. Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons: OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1).

    Science.gov (United States)

    Hewett, Sandra J; Shi, Jingxue; Gong, Yifan; Dhandapani, Krishnan; Pilbeam, Carol; Hewett, James A

    2016-12-30

    Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms. © 2016 by The American Society for Biochemistry

  13. Inhibition of CREB binding protein-beta-catenin signaling down regulates CD133 expression and activates PP2A-PTEN signaling in tumor initiating liver cancer cells.

    Science.gov (United States)

    Tang, Yuanyuan; Berlind, Joshua; Mavila, Nirmala

    2018-03-12

    The WNT-beta-catenin pathway is known to regulate cellular homeostasis during development and tissue regeneration. Activation of WNT signaling increases the stability of cytoplasmic beta-catenin and enhances its nuclear translocation. Nuclear beta-catenin function is regulated by transcriptional co-factors such as CREB binding protein (CBP) and p300. Hyper-activated WNT-beta-catenin signaling is associated with many cancers. However, its role in inducing stemness to liver cancer cells, its autoregulation and how it regulates tumor suppressor pathways are not well understood. Here we have investigated the role of CBP-beta-catenin signaling on the expression of CD133, a known stem cell antigen and PP2A-PTEN pathway in tumor initiating liver cancer cells. Human hepatoblastoma cell line HepG2 and clonally expanded CD133 expressing tumor initiating liver cells (TICs) from premalignant murine liver were used in this study. CBP-beta-catenin inhibitor ICG001 was used to target CBP-beta catenin signaling in liver cancer cells in vitro. Western blotting and real time PCR (qPCR) were used to quantify protein expression/phosphorylation and mRNA levels, respectively. CBP and CD133 gene silencing was performed by siRNA transfection. Fluorescence Activated Cell Sorting (FACS) was performed to quantify CD133 positive cells. Protein Phosphatase (PP2A) activity was measured after PP2AC immunoprecipitation. CBP inhibitor ICG001 and CBP silencing significantly reduced CD133 expression and anchorage independent growth in HepG2 and murine TICs. CD133 silencing in TICs decreased cell proliferation and expression levels of cell cycle regulatory genes, CyclinD1 and CyclinA2. ICG001 treatment and CBP silencing reduced the levels of phospho Ser380/Tyr382/383 PTEN, phospho Ser473 -AKT, Phospho- Ser552 beta-catenin in TICs. ICG001 mediated de-phosphorylation of PTEN in TICs was PP2A dependent and partly prevented by co-treatment with PP2A inhibitor okadaic acid. CBP-beta-catenin signaling

  14. Conformational control of the binding of the transactivation domain of the MLL protein and c-Myb to the KIX domain of CREB.

    Directory of Open Access Journals (Sweden)

    Elif Nihal Korkmaz

    Full Text Available The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.

  15. Prostaglandin E1 reduces the keratinocyte toxicity of sorafenib by maintaining signal transducer and activator of transcription 3 (STAT3) activity and enhancing the cAMP response element binding protein (CREB) activity.

    Science.gov (United States)

    Shichiri, Hiroaki; Yamamoto, Kazuhiro; Tokura, Maya; Ishida, Takahiro; Uda, Atsushi; Bito, Toshinori; Nishigori, Chikako; Nakagawa, Tsutomu; Hirano, Takeshi; Yano, Ikuko; Hirai, Midori

    2017-04-01

    Hand-foot skin reaction (HFSR) is a common side effect of multiple tyrosine kinase inhibitors (mTKIs). HFSR can necessitate dose reductions or interruption of therapy owing to its negative effect on the quality of life. Therefore, effective use of mTKIs requires measures to prevent HFSR. We evaluated the effect of prostaglandin E 1 (PGE 1 ) on HFSR, because PGE 1 is already used to treat bed sores and skin ulcers and has established angiogenic and antiproliferative effects in keratinocytes. We found that the pathogenesis of sorafenib-induced HFSR is characterized by a decrease in levels of a phosphorylated signal transducer and activator of transcription 3 (STAT3). We investigated the effect of PGE 1 on the sorafenib-mediated reduction in phosphorylated STAT3 levels in HaCaT human epidermal keratinocytes. In cells treated with sorafenib, phosphorylated STAT3 levels decreased in a concentration-dependent manner, and this effect was blocked in cells treated with sorafenib and PGE 1 . Furthermore, the expression of phosphorylated STAT3, the antiapoptotic proteins myeloid cell leukemia-1 (Mcl-1) and survivin decreased in cells pretreated with an inhibitor of cAMP response element binding protein (CREB). Cell viability increased in cells treated with sorafenib and PGE 1 compared with that in cells treated with sorafenib alone, and these effects were not observed in STAT3 knockdown HaCaT cells. Collectively, these findings indicate that PGE 1 blocks the inhibitory effects of sorafenib on cell growth by maintaining the activity of STAT3 and enhancing the CREB activity. Therefore, PGE 1 might represent an effective treatment for the prevention of sorafenib-induced HFSR. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Expression of cAMP-responsive element binding proteins (CREBs) in fast- and slow-twitch muscles: a signaling pathway to account for the synaptic expression of collagen-tailed subunit (ColQ) of acetylcholinesterase at the rat neuromuscular junction.

    Science.gov (United States)

    Choi, Roy C Y; Chen, Vicky P; Luk, Wilson K W; Yung, Amanda W Y; Ng, Alice H M; Dong, Tina T X; Tsim, Karl W K

    2013-03-25

    The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1a's synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

    Energy Technology Data Exchange (ETDEWEB)

    Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui; Ha, Hyunil, E-mail: hyunil74@hotmail.com; Lee, Zang Hee, E-mail: zang1959@snu.ac.kr

    2010-07-23

    Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

  18. Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus

    Science.gov (United States)

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

  19. CREB-dependent gene regulation by prion protein: impact on MMP-9 and beta-dystroglycan.

    Science.gov (United States)

    Pradines, Elodie; Loubet, Damien; Schneider, Benoît; Launay, Jean-Marie; Kellermann, Odile; Mouillet-Richard, Sophie

    2008-11-01

    Corruption of the normal function of the cellular prion protein (PrP(C)) by the scrapie isoform (PrP(Sc)) emerges as a critical causal event in Transmissible Spongiform Encaphalopathies (TSE) pathogenesis. However, PrP(C) physiological role remains unclear. By exploiting the properties of the 1C11 neuroectodermal cell line, able to convert into 1C11(5-HT) serotonergic or 1C11(NE) noradrenergic neuronal cells, we assigned a signaling function to PrP(C). Here, we establish that antibody-mediated PrP(C) ligation promotes the recruitment of the cAMP responsive element binding protein (CREB) transcription factor downstream from the MAPK ERK1/2, in 1C11 precursor cells and their 1C11(5-HT) and 1C11(NE) neuronal progenies. Whatever the differentiation state of 1C11 cells, the PrP(C)-dependent CREB activation triggers Egr-1 and c-fos transcription, two immediate early genes that relay CREB's role in cell survival and proliferation as well as in neuronal plasticity. Furthermore, in 1C11-derived neuronal cells, we draw a link between the PrP(C)-CREB coupling and a transcriptional regulation of the metalloproteinase MMP-9 and its inhibitor TIMP-1, which play pivotal roles in neuronal pathophysiology. Finally, the PrP(C)-dependent control on MMP-9 impacts on the processing of the transmembrane protein, beta-dystroglycan. Taken together, our data define molecular mechanisms that likely mirror PrP(C) ubiquitous contribution to cytoprotection and its involvement in neuronal plasticity.

  20. Chronic enhancement of CREB activity in the hippocampus interferes with the retrieval of spatial information

    OpenAIRE

    Viosca, Jose; Malleret, Gaël; Bourtchouladze, Rusiko; Benito, Eva; Vronskava, Svetlana; Kandel, Eric R.; Barco, Angel

    2009-01-01

    The activation of cAMP-responsive element-binding protein (CREB)-dependent gene expression is thought to be critical for the formation of different types of long-term memory. To explore the consequences of chronic enhancement of CREB function on spatial memory in mammals, we examined spatial navigation in bitransgenic mice that express in a regulated and restricted manner a constitutively active form of CREB, VP16-CREB, in forebrain neurons. We found that chronic enhancement of CREB activity ...

  1. Expression profile of CREB knockdown in myeloid leukemia cells

    International Nuclear Information System (INIS)

    Pellegrini, Matteo; Cheng, Jerry C; Voutila, Jon; Judelson, Dejah; Taylor, Julie; Nelson, Stanley F; Sakamoto, Kathleen M

    2008-01-01

    The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA. By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA. We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

  2. CREB: the unindicted cancer co-conspirator.

    Science.gov (United States)

    Conkright, Michael D; Montminy, Marc

    2005-09-01

    The transcription factor cAMP response-element binding protein (CREB) participates in a diverse array of cellular processes, including survival, proliferation and glucose metabolism. A new report by Shankar et al. shows that patients with acute myeloid leukemia (AML) have a greater incidence of relapse when intracellular levels of CREB are elevated. By enhancing expression of certain cell-cycle genes, CREB appears to promote growth-factor-independent proliferation and survival of myeloid cells. The results provide new insights into CREB function and suggest potential avenues for therapeutic intervention.

  3. CREB is activated by muscle injury and promotes muscle regeneration.

    Science.gov (United States)

    Stewart, Randi; Flechner, Lawrence; Montminy, Marc; Berdeaux, Rebecca

    2011-01-01

    The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration.

  4. In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac

    DEFF Research Database (Denmark)

    Kortenoeven, Marleen; Trimpert, Christiane; van den Brand, Michiel

    2012-01-01

    kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE...

  5. Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2.

    Science.gov (United States)

    Luo, Qianyi; Viste, Kristin; Urday-Zaa, Janny Concha; Senthil Kumar, Ganesan; Tsai, Wen-Wei; Talai, Afsaneh; Mayo, Kelly E; Montminy, Marc; Radhakrishnan, Ishwar

    2012-12-18

    Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

  6. CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli

    NARCIS (Netherlands)

    Barrot, Michel; Olivier, Jocelien D A; Perrotti, Linda I; DiLeone, Ralph J; Berton, Olivier; Eisch, Amelia J; Impey, Soren; Storm, Daniel R; Neve, Rachael L; Yin, Jerry C; Zachariou, Venetia; Nestler, Eric J

    2002-01-01

    The transcription factor cAMP response element (CRE)-binding protein (CREB) has been shown to regulate neural plasticity. Drugs of abuse activate CREB in the nucleus accumbens, an important part of the brain's reward pathways, and local manipulations of CREB activity have been shown to affect

  7. Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

    Science.gov (United States)

    Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

    2012-01-01

    Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

  8. Naloxone's pentapeptide binding site on filamin A blocks Mu opioid receptor-Gs coupling and CREB activation of acute morphine.

    Directory of Open Access Journals (Sweden)

    Hoau-Yan Wang

    Full Text Available Chronic morphine causes the mu opioid receptor (MOR to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Galphas and its Gbetagamma dimer. Ultra-low-dose naloxone (NLX prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA, a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 microM morphine was completely prevented by co-treatment with 100 pM NLX, (+NLX, or naltrexone (NTX, or their pentapeptide binding site (FLNA(2561-2565, which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR-FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S(133. Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS(133CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR-Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR-Gs coupling by 100 pM NLX/NTX or 10 microM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential.

  9. Naloxone's pentapeptide binding site on filamin A blocks Mu opioid receptor-Gs coupling and CREB activation of acute morphine.

    Science.gov (United States)

    Wang, Hoau-Yan; Burns, Lindsay H

    2009-01-01

    Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Galphas and its Gbetagamma dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 microM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA(2561-2565)), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR-FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S(133). Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS(133)CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR-Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR-Gs coupling by 100 pM NLX/NTX or 10 microM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential.

  10. Helical propensity in an intrinsically disordered protein accelerates ligand binding

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Dogan, Jakob; Jemth, Per

    2014-01-01

    domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure......Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation...... the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins....

  11. Cyclic AMP response element binding protein and brain-derived ...

    Indian Academy of Sciences (India)

    Madhu

    the role of CREB and BDNF in depression and as targets/mediators of antidepressant action. [Nair A and Vaidya V A 2006 Cyclic AMP response element binding protein and brain-derived neurotrophic factor: Molecules that modulate our mood?; J. Biosci. 31 423–434]. Keywords. Antidepressant; depression; hippocampus ...

  12. CREB: A Key Regulator of Normal and Neoplastic Hematopoiesis

    Directory of Open Access Journals (Sweden)

    Salemiz Sandoval

    2009-01-01

    Full Text Available The cAMP response element-binding protein (CREB is a nuclear transcription factor downstream of cell surface receptors and mitogens that is critical for normal and neoplastic hematopoiesis. Previous work from our laboratory demonstrated that a majority of patients with acute myeloid leukemia (AML and acute lymphoid leukemia (ALL overexpress CREB in the bone marrow. To understand the role of CREB in leukemogenesis, we examined the biological effect of CREB overexpression on primary leukemia cells, leukemia cell lines, and CREB overexpressing transgenic mice. Our results demonstrated that CREB overexpression leads to an increase in cellular proliferation and survival. Furthermore, CREB transgenic mice develop a myeloproliferative disorder with aberrant myelopoiesis in both the bone marrow and spleen. Additional research from other groups has shown that the expression of the cAMP early inducible repressor (ICER, a CREB repressor, is also deregulated in leukemias. And, miR-34b, a microRNA that negative regulates CREB expression, is expressed at lower levels in myeloid leukemia cell lines compared to that of healthy bone marrow. Taken together, these data suggest that CREB plays a role in cellular transformation. The data also suggest that CREB-specific signaling pathways could possibly serve as potential targets for therapeutic intervention.

  13. Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus

    Science.gov (United States)

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees (Apis mellifera) we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees’ CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus. PMID:27084927

  14. Prostaglandin E1 Attenuates Pulmonary Artery Remodeling by Activating Phosphorylation of CREB and the PTEN Signaling Pathway

    OpenAIRE

    Lai, Ying-Ju; Hsu, Hsao-Hsun; Chang, Gwo-Jyh; Lin, Shu-Hui; Chen, Wei-Jan; Huang, Chung-Chi; Pang, Jong-Hwei S.

    2017-01-01

    The depletion of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and phosphatase and tensin homolog (PTEN) is the critical mediator of pulmonary arterial hypertension (PAH). We hypothesized that the activation of phosphorylated CREB (pCREB) and PTEN could inhibit the AKT signaling pathway to attenuate pulmonary arterial remodeling in rats with monocrotaline-induced PAH. We observed decreased PTEN and pCREB in idiopathic PAH versus control tissue. We reduced PTEN ...

  15. CREB1/ATF1 activation in photoreceptor degeneration and protection.

    Science.gov (United States)

    Beltran, William A; Allore, Heather G; Johnson, Elizabeth; Towle, Virginia; Tao, Weng; Acland, Gregory M; Aguirre, Gustavo D; Zeiss, Caroline J

    2009-11-01

    The cAMP response element binding protein 1 (CREB1) and activating transcription factor 1 (ATF1) are closely related members of the bZIP superfamily of transcription factors. Both are activated in response to a wide array of stimuli, including cellular stress. This study was conducted to assess the CREB1/ATF1 pathway in photoreceptor disease and protection. The expression levels of p-CREB1, CREB1, and ATF1 were examined by immunoblot and immunohistochemistry in normal canine retina and retinas of several canine models of retinal degeneration (rcd1, rcd2, erd, prcd, XLPRA1, XLPRA2, T4R RHO). Humans retinas affected with age-related macular degeneration (AMD) were also examined. p-CREB1/ATF1 immunolabeling was assessed in normal and rcd1 dogs treated with ciliary neurotrophic factor (CNTF), to examine the effect of a neuroprotective stimulus on activation of CREB1/ATF1. Native CREB1 and ATF1 as well as phosphorylated CREB1/ATF1 was examined in normal canine retina by immunoblot. The p-CREB1 antibody identified phosphorylated CREB1 and ATF1 and labeled the inner retina only in normal dogs. In degenerate canine and human retinas, strong immunolabeling appeared in rod and cone photoreceptors, indicating increased expression of native CREB1 and ATF1, as well as increased phosphorylation of these proteins. Retinal protection by CNTF in rcd1 dogs was accompanied by a significant increase in the number of p-CREB1/ATF1-labeled photoreceptor nuclei. Positive association of CREB1/ATF1 phosphorylation with photoreceptor protection suggests that it may contribute to an innate protective response. These data identify a signaling mechanism in rods and cones of potential importance for therapies of RP and AMD.

  16. Prenatal auditory stimulation alters the levels of CREB mRNA, p-CREB and BDNF expression in chick hippocampus.

    Science.gov (United States)

    Chaudhury, Sraboni; Wadhwa, Shashi

    2009-10-01

    Prenatal auditory stimulation influences the development of the chick auditory pathway and the hippocampus showing an increase in various morphological parameters as well as expression of calcium-binding proteins. Calcium regulates the activity of cyclic adenosine monophosphate-response element binding (CREB) protein. CREB is known to play a role in development, undergo phosphorylation with neural activity as well as regulate transcription of BDNF. BDNF is important for the survival of neurons and regulates synaptic strength. Hence in the present study, we have evaluated the levels of CREB mRNA and protein along with p-CREB protein as well as BDNF mRNA and protein levels in the chick hippocampus at embryonic days (E) 12, E16, E20 and post-hatch day (PH) 1 following activation by prenatal auditory stimulation. Fertilized eggs were exposed to species-specific sound or sitar music (frequency range: 100-6300Hz) at 65dB levels for 15min/h over 24h from E10 till hatching. The control chick hippocampus showed higher CREB mRNA and p-CREB protein in the early embryonic stages, which later decline whereas BDNF mRNA and BDNF protein levels increase until PH1. The CREB mRNA and p-CREB protein were significantly increased at E12, E16 and PH1 in the auditory stimulated groups as compared to control group. A significant increase in the level of BDNF mRNA was observed from E12 and the protein expression from E16 onwards in both auditory stimulated groups. Therefore, enhanced phosphorylation of CREB during development following prenatal sound stimulation may be responsible for cell survival. Increased levels of p-CREB again at PH1 may trigger synthesis of proteins necessary for synaptic plasticity. Further, the increased levels of BDNF may also help in regulating synaptic plasticity.

  17. CREB Overexpression Ameliorates Age-related Behavioral and Biophysical Deficits

    Science.gov (United States)

    Yu, Xiao-Wen

    Age-related cognitive deficits are observed in both humans and animals. Yet, the molecular mechanisms underlying these deficits are not yet fully elucidated. In aged animals, a decrease in intrinsic excitability of pyramidal neurons from the CA1 sub-region of hippocampus is believed to contribute to age-related cognitive impairments, but the molecular mechanism(s) that modulate both these factors has yet to be identified. Increasing activity of the transcription factor cAMP response element-binding protein (CREB) in young adult rodents has been shown to facilitate cognition, and increase intrinsic excitability of their neurons. However, how CREB changes with age, and how that impacts cognition in aged animals, is not clear. Therefore, we first systematically characterized age- and training-related changes in CREB levels in dorsal hippocampus. At a remote time point after undergoing behavioral training, levels of total CREB and activated CREB (phosphorylated at S133, pCREB) were measured in both young and aged rats. We found that pCREB, but not total CREB was significantly reduced in dorsal CA1 of aged rats. Importantly, levels of pCREB were found to be positively correlated with short-term spatial memory in both young and aged rats i.e. higher pCREB in dorsal CA1 was associated with better spatial memory. These findings indicate that an age-related deficit in CREB activity may contribute to the development of age-related cognitive deficits. However, it was still unclear if increasing CREB activity would be sufficient to ameliorate age-related cognitive, and biophysical deficits. To address this question, we virally overexpressed CREB in CA1, where we found the age-related deficit. Young and aged rats received control or CREB virus, and underwent water maze training. While control aged animals exhibited deficits in long-term spatial memory, aged animals with CREB overexpression performed at levels comparable to young animals. Concurrently, aged neurons

  18. Inhibition of DNA binding activity of cAMP response element-binding protein by 1,2-naphthoquinone through chemical modification of Cys-286.

    Science.gov (United States)

    Endo, Akiko; Sumi, Daigo; Iwamoto, Noriko; Kumagai, Yoshito

    2011-07-15

    1,2-Naphthoquinone (1,2-NQ) is an atmospheric electrophile that reacts covalently with protein thiols. Our previous study revealed that exposure of bovine aortic endothelial cells to 1,2-NQ causes covalent modification of cAMP response element-binding protein (CREB), thereby inhibiting its DNA binding activity and substantial gene expression of B-cell lymphoma-2 (Bcl-2) that is regulated by this transcription factor. In this study, we identified the modification sites of CREB that are associated with the decreased transcriptional activity. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis indicated that three amino acids (Cys-286, Lys-290, and Lys-319) were irreversibly modified by 1,2-NQ. Mutational analysis revealed that electrophilic modification of Cys-286, but not the other two amino acids, at the DNA binding domain is essential for the reduced CREB activity. Substitution of Cys-286 with tryptophan (C286W), which mimics CREB modification by 1,2-NQ, supported this notion. These results suggest that the covalent interaction of CREB with 1,2-NQ through Cys-286 blocks the DNA binding activity of CREB, resulting in the repression of CREB-regulated genes. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Role of a transcription factor (CREB) in memory processes.

    Science.gov (United States)

    De Luca, A; Giuditta, A

    1997-01-01

    Memory storage includes a short-term phase (STM) which requires the phosphorylation of pre-existing proteins, and a long-term phase (LTM) which needs the novel synthesis of RNA and proteins. Cyclic AMP and a specific transcription factor (cAMP response element binding protein or CREB) play a central role in the formation of LTM in aplysia, drosophila and mice. Following its phosphorylation by protein kinase A, CREB binds to the enhancer element CRE which is located in the upstream region of cAMP-responsive genes, thus triggering transcription. Some of the newly-synthesized proteins are additional transcription factors that ultimately give rise to the activation of late response genes, whose products are responsible for the modification of synaptic efficacy leading to LTM. In aplysia, CREB activation has been interfered with by microinjection of CRE containing oligonucleotides into cultured neurons. Under these conditions LTM is blocked while STM remains unchanged. In drosophila, CREB function has been disrupted using a reverse genetic approach. Thus, LTM has been specifically blocked by the induced expression of a CREB repressor isoform, and enhanced by the induced expression of an activator isoform. In mouse, the role of CREB has been confirmed by behavioural analyses of a knock-out line with a targeted mutation in the CREB gene. In these mutants, learning and STM are normal, whereas LTM is disrupted. On the whole, the data suggest that encoding of long term memories involve highly conserved molecular mechanisms.

  20. Protein kinases mediate increment of the phosphorylation of cyclic AMP -responsive element binding protein in spinal cord of rats following capsaicin injection

    Directory of Open Access Journals (Sweden)

    Li Junfa

    2005-09-01

    Full Text Available Abstract Background Strong noxious stimuli cause plastic changes in spinal nociceptive neurons. Intracellular signal transduction pathways from cellular membrane to nucleus, which may further regulate gene expression by critical transcription factors, convey peripheral stimulation. Cyclic AMP-responsive element binding protein (CREB is a well-characterized stimulus-induced transcription factor whose activation requires phosphorylation of the Serine-133 residue. Phospho-CREB can further induce gene transcription and strengthen synaptic transmission by the activation of the protein kinase cascades. However, little is known about the mechanisms by which CREB phosphorylation is regulated by protein kinases during nociception. This study was designed to use Western blot analysis to investigate the role of mitogen-activated protein (MAP/extracellular signal-regulated kinase (ERK kinase (MEK 1/2, PKA and PKC in regulating the phosphorylation of CREB in the spinal cord of rats following intraplantar capsaicin injection. Results We found that capsaicin injection significantly increased the phosphorylation level of CREB in the ipsilateral side of the spinal cord. Pharmacological manipulation of MEK 1/2, PKA and PKC with their inhibitors (U0126, H89 and NPC 15473, respectively significantly blocked this increment of CREB phosphorylation. However, the expression of CREB itself showed no change in any group. Conclusion These findings suggest that the activation of intracellular MAP kinase, PKA and PKC cascades may contribute to the regulation of phospho-CREB in central nociceptive neurons following peripheral painful stimuli.

  1. Chronic Enhancement of CREB Activity in the Hippocampus Interferes with the Retrieval of Spatial Information

    Science.gov (United States)

    Viosca, Jose; Malleret, Gael; Bourtchouladze, Rusiko; Benito, Eva; Vronskava, Svetlana; Kandel, Eric R.; Barco, Angel

    2009-01-01

    The activation of cAMP-responsive element-binding protein (CREB)-dependent gene expression is thought to be critical for the formation of different types of long-term memory. To explore the consequences of chronic enhancement of CREB function on spatial memory in mammals, we examined spatial navigation in bitransgenic mice that express in a…

  2. Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.

    Science.gov (United States)

    Zhang, Xinmin; Odom, Duncan T; Koo, Seung-Hoi; Conkright, Michael D; Canettieri, Gianluca; Best, Jennifer; Chen, Huaming; Jenner, Richard; Herbolsheimer, Elizabeth; Jacobsen, Elizabeth; Kadam, Shilpa; Ecker, Joseph R; Emerson, Beverly; Hogenesch, John B; Unterman, Terry; Young, Richard A; Montminy, Marc

    2005-03-22

    Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.

  3. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals.

    Science.gov (United States)

    Altarejos, Judith Y; Montminy, Marc

    2011-03-01

    The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator-co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

  4. CREB inhibits AP-2alpha expression to regulate the malignant phenotype of melanoma.

    Directory of Open Access Journals (Sweden)

    Vladislava O Melnikova

    2010-08-01

    Full Text Available The loss of AP-2alpha and increased activity of cAMP-responsive element binding (CREB protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2alpha during melanoma progression remains unknown.Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2alpha protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2alpha. Loss of AP-2alpha expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2alpha promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2alpha expression following CREB silencing increases endogenous p21(Waf1 and decreases MCAM/MUC18, both known to be downstream target genes of AP-2alpha involved in melanoma progression.Since AP-2alpha regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21(Waf1, our data identified CREB as a major regulator of the malignant melanoma phenotype.

  5. Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB

    Science.gov (United States)

    Zhang, Yanling.; Zhen, Wei.; Maechler, Pierre; Liu, Dongmin

    2013-01-01

    Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of T2D. Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis, and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of anti-apoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and PDX-1 expression. Chronic hyperlipidemia significantly diminished cAMP production, PKA activation, and CREB phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48 h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol–stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade. PMID:22819546

  6. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    International Nuclear Information System (INIS)

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li; Li Junfa

    2006-01-01

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning

  7. Adipocyte CREB promotes insulin resistance in obesity.

    Science.gov (United States)

    Qi, Ling; Saberi, Maziyar; Zmuda, Erik; Wang, Yiguo; Altarejos, Judith; Zhang, Xinmin; Dentin, Renaud; Hedrick, Susie; Bandyopadhyay, Gautam; Hai, Tsonwin; Olefsky, Jerry; Montminy, Marc

    2009-03-01

    Increases in adiposity trigger metabolic and inflammatory changes that interfere with insulin action in peripheral tissues, culminating in beta cell failure and overt diabetes. We found that the cAMP Response Element Binding protein (CREB) is activated in adipose cells under obese conditions, where it promotes insulin resistance by triggering expression of the transcriptional repressor ATF3 and thereby downregulating expression of the adipokine hormone adiponectin as well as the insulin-sensitive glucose transporter 4 (GLUT4). Transgenic mice expressing a dominant-negative CREB transgene in adipocytes displayed increased whole-body insulin sensitivity in the contexts of diet-induced and genetic obesity, and they were protected from the development of hepatic steatosis and adipose tissue inflammation. These results indicate that adipocyte CREB provides an early signal in the progression to type 2 diabetes.

  8. Increasing levels of wild-type CREB up-regulates several activity-regulated inhibitor of death (AID genes and promotes neuronal survival

    Directory of Open Access Journals (Sweden)

    Tan Yan-Wei

    2012-05-01

    Full Text Available Abstract Background CREB (cAMP-response element binding protein is the prototypical signal-regulated transcription factor. In neurons, it is the target of the synaptic activity-induced nuclear calcium-calcium/calmodulin dependent protein kinase (CaMK IV signaling pathway that controls the expression of genes important for acquired neuroprotection as well as other long-lasting adaptive processes in the nervous system. The function of CREB as a transcriptional activator is controlled by its phosphorylation on serine 133, which can be catalyzed by CaMKIV and leads to the recruitment of the co-activator, CREB binding protein (CBP. Activation of CBP function by nuclear calcium-CaMKIV signaling is a second regulatory step required for CREB/CBP-mediated transcription. Results Here we used recombinant adeno-associated virus (rAAV to increase the levels of wild type CREB or to overexpress a mutant version of CREB (mCREB containing a serine to alanine mutation at position amino acid 133 in mouse hippocampal neurons. Increasing the levels of CREB was sufficient to boost neuroprotective activity even under basal conditions (i.e., in the absence of stimulation of synaptic activity. In contrast, overexpression of mCREB increased cell death. The ratio of phospho(serine 133CREB to CREB immunoreactivity in unstimulated hippocampal neurons was similar for endogenous CREB and overexpressed wild type CREB and, as expected, dramatically reduced for overexpressed mCREB. A gene expression analysis revealed that increased expression of CREB but not that of mCREB in hippocampal neurons led to elevated expression levels of bdnf as well as that of several members of a previously characterized set of Activity-regulated Inhibitor of Death (AID genes, which include atf3, btg2, gadd45β, and gadd45γ. Conclusions Our findings indicate that the expression levels of wild type CREB are a critical determinant of the ability of hippocampal neurons to survive harmful conditions

  9. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  10. CREB promotes laryngeal cancer cell migration via MYCT1/NAT10 axis

    Directory of Open Access Journals (Sweden)

    Zhang ZX

    2018-03-01

    Full Text Available Zhao-Xiong Zhang,1 Wan-Ni Zhang,1 Yuan-Yuan Sun,1 Yun-Hui Li,2 Zhen-Ming Xu,3 Wei-Neng Fu1 1Department of Medical Genetics, China Medical University, Shenyang, People’s Republic of China; 2Department of Laboratory Medicine, No 202 Hospital of PLA, Shenyang, People’s Republic of China; 3Department of Otolaryngology, No 463 Hospital of PLA, Shenyang, People’s Republic of China Purpose: CREB, MYCY1 and NAT10 are involved in cancer cell migration. However, the relationship between these three proteins and their role in laryngeal cancer cell migration remains unknown. Methods: Transient gene transfection was performed in laryngeal cancer cells. Bioinformatics analysis was used to predict the binding of CREB to MYCT1 promoter. Binding of CREB to the promoter of MYCT1 was monitored by luciferase reporter assay and chromatin immunoprecipitation method in vitro and in vivo, respectively. Real-time RT-PCR and Western bolt were applied to detect gene transcription and translation levels, respectively. Laryngeal cancer cell migration was assayed by transwell cham­ber experiment. Results: CREB protein expression was significantly up-regulated in laryngeal cancer tissues and associated with cancer differentiation, tumor stage, and lymphatic metasta­sis. CREB inhibits MYCT1 expression by direct binding to its promoter. Meanwhile, MYCT1 has a negative impact on the NAT10 gene expression. Furthermore, CREB promotes NAT10 expression via down-regulating the MYCT1 gene expression. In addition, contrary to MYCT1, CREB and NAT10 enhanced laryngeal cancer cell migration. MYCT1 and NAT10 significantly rescued the effects of CREB and MYCT1 on Hep2 cell migration, respectively. Conclusion: CREB promotes laryngeal cancer cell migration via MYCT1/NAT10 axis, suggesting that CREB might be a potential prognostic marker in laryngeal cancer. Keywords: laryngeal cancer, CREB, MYCT1, NAT10, migration

  11. Genetic evidence for a role of CREB in sustained cortical arousal.

    Science.gov (United States)

    Graves, Laurel A; Hellman, Kevin; Veasey, Sigrid; Blendy, Julie A; Pack, Allan I; Abel, Ted

    2003-08-01

    The cyclic AMP-response element binding protein (CREB) is an activity-dependent transcription factor important for synaptic plasticity and memory storage. Levels of phosphorylated CREB within the cortex are higher in waking than in sleep, suggesting that CREB plays a role in sleep/wake regulation in mammals. We tested the hypothesis that CREB is critical for sleep/wake regulation by examining behavioral state parameters in mice lacking the alpha and Delta isoforms of CREB. Over 24 h, time spent awake was significantly decreased in CREB alphaDelta mutant mice by approximately 100 min, and time spent in nonrapid eye movement sleep (NREM) sleep was increased correspondingly. Wake and REM sleep periods were shorter in CREB alphaDelta mice, and CREB alphaDelta mice had decreased levels of -activity during wake and REM sleep, consistent with an impairment in the ability to maintain an activated electroencephalogram. These results suggest that the CREB protein contributes to the mechanisms by which wakefulness is maintained and demonstrate that specific genetic alterations in species as diverse as Drosophila and mice produce similar phenotypes in arousal and wakefulness.

  12. Mechanisms Underlying the Antidepressant Response of Acupuncture via PKA/CREB Signaling Pathway.

    Science.gov (United States)

    Jiang, Huili; Zhang, Xuhui; Wang, Yu; Zhang, Huimin; Li, Jing; Yang, Xinjing; Zhao, Bingcong; Zhang, Chuntao; Yu, Miao; Xu, Mingmin; Yu, Qiuyun; Liang, Xingchen; Li, Xiang; Shi, Peng; Bao, Tuya

    2017-01-01

    Protein kinase A (PKA)/cAMP response element-binding (CREB) protein signaling pathway, contributing to impaired neurogenesis parallel to depressive-like behaviors, has been identified as the crucial factor involved in the antidepressant response of acupuncture. However, the molecular mechanisms associated with antidepressant response of acupuncture, neurogenesis, and depressive-like behaviors ameliorating remain unexplored. The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor H89 in in vivo experiments. Our results indicated that the expression of hippocampal PKA- α and p-CREB was significantly downregulated by chronic unpredicted mild stress (CUMS) procedures. Importantly, acupuncture reversed the downregulation of PKA- α and p-CREB. The expression of PKA- α was upregulated by fluoxetine, but not p-CREB. No significant difference was found between Acu and FLX groups on the expression of PKA- α and p-CREB. Interestingly, H89 inhibited the effects of acupuncture or fluoxetine on upregulating the expression of p-CREB, but not PKA- α . There was no significant difference in expression of CREB among the groups. Conclusively, our findings further support the hypothesis that acupuncture could ameliorate depressive-like behaviors by regulating PKA/CREB signaling pathway, which might be mainly mediated by regulating the phosphorylation level of CREB.

  13. Acupuncture Activates ERK-CREB Pathway in Rats Exposed to Chronic Unpredictable Mild Stress

    Directory of Open Access Journals (Sweden)

    Jun Lu

    2013-01-01

    Full Text Available Extracellular signal-regulated kinase (ERK-cAMP response element binding protein (CREB signal pathway has been implicated in the pathogenesis of depression. There is growing evidence that acupuncture in traditional Chinese medicine has antidepressant-like effect. However, the effect of acupuncture on ERK-CREB pathway remains unknown. In our study, the antidepressant-like effect of acupuncture treatment was measured by sucrose intake test and open field test in rats exposed to chronic unpredictable mild stress (CUMS for 4 weeks. The protein levels of ERK1/2, CREB, phosphorylated ERK1/2 (p-ERK1/2, and phosphorylated CREB (p-CREB in the hippocampus (HP and prefrontal cortex (PFC were examined by Western blot analysis. Our results showed that CUMS rats exhibited the reduction in behavioral activities, whereas acupuncture stimulation at acupoints Baihui (Du20 and Neiguan (PC6 reversed the behavioral deficit. In addition, exposure to CUMS resulted in the decrease of p-ERK1/2 and p-CREB in the HP and PFC. Acupuncture increased the ratio of p-ERK1/2 to ERK1/2 and the ratio of p-CREB to CREB in the HP and PFC. Our study suggested that one potential way, by which acupuncture had antidepressant-like effect, might be mediated by activating the ERK-CREB pathway in the brain.

  14. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  15. Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

    Directory of Open Access Journals (Sweden)

    Dmitry Akhmedov

    Full Text Available The cAMP response element binding protein (CREB is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc. cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

  16. CREB Selectively Controls Learning-Induced Structural Remodeling of Neurons

    Science.gov (United States)

    Middei, Silvia; Spalloni, Alida; Longone, Patrizia; Pittenger, Christopher; O'Mara, Shane M.; Marie, Helene; Ammassari-Teule, Martine

    2012-01-01

    The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons…

  17. The mammalian INDY homolog is induced by CREB in a rat model of type 2 diabetes.

    Science.gov (United States)

    Neuschäfer-Rube, Frank; Lieske, Stefanie; Kuna, Manuela; Henkel, Janin; Perry, Rachel J; Erion, Derek M; Pesta, Dominik; Willmes, Diana M; Brachs, Sebastian; von Loeffelholz, Christian; Tolkachov, Alexander; Schupp, Michael; Pathe-Neuschäfer-Rube, Andrea; Pfeiffer, Andreas F H; Shulman, Gerald I; Püschel, Gerhard P; Birkenfeld, Andreas L

    2014-03-01

    Reduced expression of the INDY (I'm not dead yet) tricarboxylate carrier increased the life span in different species by mechanisms akin to caloric restriction. Mammalian INDY homolog (mIndy, SLC13A5) gene expression seems to be regulated by hormonal and/or nutritional factors. The underlying mechanisms are still unknown. The current study revealed that mIndy expression and [(14)C]-citrate uptake was induced by physiological concentrations of glucagon via a cAMP-dependent and cAMP-responsive element-binding protein (CREB)-dependent mechanism in primary rat hepatocytes. The promoter sequence of mIndy located upstream of the most frequent transcription start site was determined by 5'-rapid amplification of cDNA ends. In silico analysis identified a CREB-binding site within this promoter fragment of mIndy. Functional relevance for the CREB-binding site was demonstrated with reporter gene constructs that were induced by CREB activation when under the control of a fragment of a wild-type promoter, whereas promoter activity was lost after site-directed mutagenesis of the CREB-binding site. Moreover, CREB binding to this promoter element was confirmed by chromatin immunoprecipitation in rat liver. In vivo studies revealed that mIndy was induced in livers of fasted as well as in high-fat-diet-streptozotocin diabetic rats, in which CREB is constitutively activated. mIndy induction was completely prevented when CREB was depleted in these rats by antisense oligonucleotides. Together, these data suggest that mIndy is a CREB-dependent glucagon target gene that is induced in fasting and in type 2 diabetes. Increased mIndy expression might contribute to the metabolic consequences of diabetes in the liver.

  18. The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase.

    Science.gov (United States)

    Namkoong, Sim; Lee, Kang Il; Lee, Jin I; Park, Rackhyun; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

    2015-01-01

    The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

  19. Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

    Directory of Open Access Journals (Sweden)

    Hisayo Sadamoto

    2010-05-01

    Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

  20. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  1. Roles of p300 and cyclic adenosine monophosphate response element binding protein in high glucose-induced hypoxia-inducible factor 1α inactivation under hypoxic conditions.

    Science.gov (United States)

    Ding, Lingtao; Yang, Minlie; Zhao, Tianlan; Lv, Guozhong

    2017-05-01

    Given the high prevalence of diabetes and burn injuries worldwide, it is essential to dissect the underlying mechanism of delayed burn wound healing in diabetes patients, especially the high glucose-induced hypoxia-inducible factor 1 (HIF-1)-mediated transcription defects. Human umbilical vein endothelial cells were cultured with low or high concentrations of glucose. HIF-1α-induced vascular endothelial growth factor (VEGF) transcription was measured by luciferase assay. Immunofluorescence staining was carried out to visualize cyclic adenosine monophosphate response element binding protein (CREB) localization. Immunoprecipitation was carried out to characterize the association between HIF-1α/p300/CREB. To test whether p300, CREB or p300+CREB co-overexpression was sufficient to rescue the HIF-1-mediated transcription defect after high glucose exposure, p300, CREB or p300+CREB co-overexpression were engineered, and VEGF expression was quantified. Finally, in vitro angiogenesis assay was carried out to test whether the high glucose-induced angiogenesis defect is rescuable by p300 and CREB co-overexpression. Chronic high glucose treatment resulted in impaired HIF-1-induced VEGF transcription and CREB exclusion from the nucleus. P300 or CREB overexpression alone cannot rescue high glucose-induced HIF-1α transcription defects. In contrast, co-overexpression of p300 and CREB dramatically ameliorated high glucose-induced impairment of HIF-1-mediated VEGF transcription, as well as in vitro angiogenesis. Finally, we showed that co-overexpression of p300 and CREB rectifies the dissociation of HIF-1α-p300-CREB protein complex in chronic high glucose-treated cells. Both p300 and CREB are required for the function integrity of HIF-1α transcription machinery and subsequent angiogenesis, suggesting future studies to improve burn wound healing might be directed to optimization of the interaction between p300, CREB and HIF-1α. © 2016 The Authors. Journal of Diabetes

  2. Viral Vector Induction of CREB Expression in the Periaqueductal Gray Induces a Predator Stress-Like Pattern of Changes in pCREB Expression, Neuroplasticity, and Anxiety in Rodents

    Directory of Open Access Journals (Sweden)

    Robert Adamec

    2009-01-01

    Full Text Available Predator stress is lastingly anxiogenic. Phosphorylation of CREB to pCREB (phosphorylated cyclic AMP response element binding protein is increased after predator stress in fear circuitry, including in the right lateral column of the PAG (periaqueductal gray. Predator stress also potentiates right but not left CeA-PAG (central amygdala-PAG transmission up to 12 days after stress. The present study explored the functional significance of pCREB changes by increasing CREB expression in non-predator stressed rats through viral vectoring, and assessing the behavioral, electrophysiological and pCREB expression changes in comparison with handled and predator stressed controls. Increasing CREB expression in right PAG was anxiogenic in the elevated plus maze, had no effect on risk assessment, and increased acoustic startle response while delaying startle habituation. Potentiation of the right but not left CeA-PAG pathway was also observed. pCREB expression was slightly elevated in the right lateral column of the PAG, while the dorsal and ventral columns were not affected. The findings of this study suggest that by increasing CREB and pCREB in the right lateral PAG, it is possible to produce rats that exhibit behavioral, brain, and molecular changes that closely resemble those seen in predator stressed rats.

  3. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature. The grids were subsequently .... and inhibition by GAGs and DMA were determined on polystyrene wells of microtitre plates (Costar, ... for binding inhibition assays was carried out by mixing equal volumes of the conjugate and the inhibitor at ...

  4. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity ... site is seen to correspond to the carbohydrate-binding site in E-selectin, which has similarity in the ... adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature.

  5. Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

    Science.gov (United States)

    Rodón, Laura; Gonzàlez-Juncà, Alba; Inda, María del Mar; Sala-Hojman, Ada; Martínez-Sáez, Elena; Seoane, Joan

    2014-10-01

    In advanced cancer, including glioblastoma, the TGFβ pathway acts as an oncogenic factor. Some tumors exhibit aberrantly high TGFβ activity, and the mechanisms underlying this phenomenon are not well understood. We have observed that TGFβ can induce TGFβ2, generating an autocrine loop leading to aberrantly high levels of TGFβ2. We identified cAMP-responsive element-binding protein 1 (CREB1) as the critical mediator of the induction of TGFβ2 by TGFβ. CREB1 binds to the TGFB2 gene promoter in cooperation with SMAD3 and is required for TGFβ to activate transcription. Moreover, the PI3K-AKT and RSK pathways regulate the TGFβ2 autocrine loop through CREB1. The levels of CREB1 and active phosphorylated CREB1 correlate with TGFβ2 in glioblastoma. In addition, using patient-derived in vivo models of glioblastoma, we found that CREB1 levels determine the expression of TGFβ2. Our results show that CREB1 can be considered a biomarker to stratify patients for anti-TGFβ treatments and a therapeutic target in glioblastoma. TGFβ is considered a promising therapeutic target, and several clinical trials using TGFβ inhibitors are generating encouraging results. Here, we discerned the molecular mechanisms responsible for the aberrantly high levels of TGFβ2 found in certain tumors, and we propose biomarkers to predict the clinical response to anti-TGFβ therapies. ©2014 American Association for Cancer Research.

  6. TORCs: transducers of regulated CREB activity.

    Science.gov (United States)

    Conkright, Michael D; Canettieri, Gianluca; Screaton, Robert; Guzman, Ernesto; Miraglia, Loren; Hogenesch, John B; Montminy, Marc

    2003-08-01

    The cAMP responsive factor CREB stimulates gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator CBP. In certain cell types, CREB also functions as a constitutive activator, although the underlying mechanisms are not understood. Here, we characterize a conserved family of coactivators, designated TORCs, for Transducers of Regulated CREB activity, that enhances CRE-dependent transcription via a phosphorylation-independent interaction with the bZIP DNA binding/dimerization domain of CREB. TORC recruitment does not appear to modulate CREB DNA binding activity, but rather enhances the interaction of CREB with the TAF(II)130 component of TFIID following its recruitment to the promoter. Remarkably, in certain mucoepidermoid carcinomas, a chromosomal translocation fuses the CREB binding domain of TORC1 to the Notch coactivator Mastermind (MAML2). As expression of the TORC1-MAML2 chimera strongly induced target gene expression via CREB, our results reveal a mechanism by which CREB stimulates transcription in normal and transformed cells.

  7. Gene regulation mediated by interaction between HTLV-1 promoter elements and transcription factors Tax and CREB.

    Science.gov (United States)

    Goren, I; Tavor, E; Honigman, A

    1999-04-10

    In this work we examine the role of three genetic control components in the regulation of HTLV-1 transcription: cyclic AMP-responsive element (CRE)-binding protein (CREB), the HTLV-1 trans-activator Tax, and the three Tax-responsive elements (TREs). We demonstrate that the in vivo efficiency of the HTLV-1 promoter basal expression in cell culture depends on the spacing between the three TRE elements, located at the HTLV-1 LTR (long terminal repeat), whereas the level of transcription activation mediated by Tax is affected by the number of TREs. In the presence of only one TRE, the enhancement of expression by Tax is affected by the distance between the single TRE and the transcription start site. Following CREB binding to the LTR, additional DNase I hypersensitive sites are generated in the region between the two distal TREs (I and II), while in the presence of Tax, such sites are generated also in the region between TREs II and III. Neither cooperative binding of CREB to the TREs nor preferential binding of CREB to a particular TRE was observed. Tax binding to the CREB/TRE complex does not change the DNase I protection pattern. Taken together, these results suggest that the basal CREB-mediated transcription is determined by the number and the position of the viral TREs relative to each other. Tax protein stabilizes the protein/DNA complex and suppresses the spacing limitations, probably by bridging between the CREB/TRE complexes and the basal initiation transcription complex. Copyright 1999 Academic Press.

  8. Prostaglandin E1 Attenuates Pulmonary Artery Remodeling by Activating Phosphorylation of CREB and the PTEN Signaling Pathway.

    Science.gov (United States)

    Lai, Ying-Ju; Hsu, Hsao-Hsun; Chang, Gwo-Jyh; Lin, Shu-Hui; Chen, Wei-Jan; Huang, Chung-Chi; Pang, Jong-Hwei S

    2017-08-30

    The depletion of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and phosphatase and tensin homolog (PTEN) is the critical mediator of pulmonary arterial hypertension (PAH). We hypothesized that the activation of phosphorylated CREB (pCREB) and PTEN could inhibit the AKT signaling pathway to attenuate pulmonary arterial remodeling in rats with monocrotaline-induced PAH. We observed decreased PTEN and pCREB in idiopathic PAH versus control tissue. We reduced PTEN using small interfering RNA in human control pulmonary arterial smooth muscle cells (PASMCs) and observed an increase in pAKT. Consistent with PTEN knockdown in PASMCs, prostaglandin E1 (PGE1) induced pCREB expression to stimulate PTEN protein expression and inhibited pAKT in a time- and dose-dependent manner. The enhanced proliferation and migration of PASMCs following PTEN knockdown were significantly inhibited by PGE1 treatment. The PGE1-induced elevation of PTEN expression in PTEN-depleted PASMCs was decreased by the application of a PKA inhibitor and a CBP-CREB interaction inhibitor. Supplementation with a novel emulsion composition comprising PGE1 in rats with monocrotaline-induced PAH prevented pulmonary arterial remodeling and improved hemodynamics via the induced expression of PTEN. We conclude that PGE1 recruits pCREB/PTEN to decrease the migration and proliferation of PASMCs associated with PAH. This finding elucidates a relevant underlying mechanism of the PGE1/CREB/PTEN signaling pathway to prevent progressive PAH.

  9. Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Satoshi Suo

    Full Text Available Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

  10. CREB Regulates Experience-Dependent Spine Formation and Enlargement in Mouse Barrel Cortex

    Directory of Open Access Journals (Sweden)

    Annabella Pignataro

    2015-01-01

    Full Text Available Experience modifies synaptic connectivity through processes that involve dendritic spine rearrangements in neuronal circuits. Although cAMP response element binding protein (CREB has a key function in spines changes, its role in activity-dependent rearrangements in brain regions of rodents interacting with the surrounding environment has received little attention so far. Here we studied the effects of vibrissae trimming, a widely used model of sensory deprivation-induced cortical plasticity, on processes associated with dendritic spine rearrangements in the barrel cortex of a transgenic mouse model of CREB downregulation (mCREB mice. We found that sensory deprivation through prolonged whisker trimming leads to an increased number of thin spines in the layer V of related barrel cortex (Contra in wild type but not mCREB mice. In the barrel field controlling spared whiskers (Ipsi, the same trimming protocol results in a CREB-dependent enlargement of dendritic spines. Last, we demonstrated that CREB regulates structural rearrangements of synapses that associate with dynamic changes of dendritic spines. Our findings suggest that CREB plays a key role in dendritic spine dynamics and synaptic circuits rearrangements that account for new brain connectivity in response to changes in the environment.

  11. A CREB-Sirt1-Hes1 Circuitry Mediates Neural Stem Cell Response to Glucose Availability

    Directory of Open Access Journals (Sweden)

    Salvatore Fusco

    2016-02-01

    Full Text Available Summary: Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1 is modulated in neural stem and progenitor cells (NSCs by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein and Sirt-1 (Sirtuin 1, two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis. : Using a combination of in vitro and in vivo studies, Fusco et al. find that excess glucose impairs the self-renewal capacity of neural stem cells through a molecular circuit that involves the transcription factor CREB and Sirtuin 1. The authors suggest that this circuitry may link nutrient excess with neurodegeneration and brain aging. Keywords: neural stem cells, adult neurogenesis, CREB, Sirt-1, nutrients, metabolism, diabetes

  12. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  13. Glucagon-CREB/CRTC2 Signaling Cascade Regulates Hepatic BMAL1 Protein*

    Science.gov (United States)

    Sun, Xiujie; Dang, Fabin; Zhang, Deyi; Yuan, Yuan; Zhang, Cui; Wu, Yuting; Wang, Yiguo; Liu, Yi

    2015-01-01

    Energy metabolism follows a diurnal pattern responding to the cycles of light and food exposures. Although food availability is a potent synchronizer of peripheral circadian clock in mammals, the underlying mechanism remains elusive. Here, we found that the temporal signals of fasting and refeeding hormones regulate the transcription of Bmal1, a key transcription activator of molecular clock, in the liver. During fasting, glucagon, a major fasting hormone, activates CREB/CRTC2 transcriptional complex that is recruited to Bmal1 promoter to induce its expression. Furthermore, we showed that CRTC2 is required for basal transcriptional regulation of Bmal1 by experiments using either adenovirus-mediated CRTC2 RNAi knockdown or primary Crtc2 null hepatocytes. On the other hand, insulin suppresses fasting-induced Bmal1 expression by inhibiting CRTC2 activity after refeeding. Taken together, our results indicate CRTC2 as a key component of the circadian oscillator that integrates the mammalian clock and energy metabolism. PMID:25480789

  14. IGF-1 protects cardiac myocytes from hyperosmotic stress-induced apoptosis via CREB

    International Nuclear Information System (INIS)

    Maldonado, Carola; Cea, Paola; Adasme, Tatiana; Collao, Andres; Diaz-Araya, Guillermo; Chiong, Mario; Lavandero, Sergio

    2005-01-01

    Hyperosmotic stress stimulates a rapid and pronounced apoptosis in cardiac myocytes which is attenuated by insulin-like growth factor-1 (IGF-1). Because in these cells IGF-1 induces intracellular Ca 2+ increase, we assessed whether the cyclic AMP response element-binding protein (CREB) is activated by IGF-1 through Ca 2+ -dependent signalling pathways. In cultured cardiac myocytes, IGF-1 induced phosphorylation (6.5 ± 1.0-fold at 5 min), nuclear translocation (30 min post-stimulus) and DNA binding activity of CREB. IGF-1-induced CREB phosphorylation was mediated by MEK1/ERK, PI3-K, p38-MAPK, as well as Ca 2+ /calmodulin kinase and calcineurin. Exposure of cardiac myocytes to hyperosmotic stress (sorbitol 600 mOsm) decreased IGF-1-induced CREB activation Moreover, overexpression of a dominant negative CREB abolished the anti-apoptotic effects of IGF-1. Our results suggest that IGF-1 activates CREB through a complex signalling pathway, and this transcription factor plays an important role in the anti-apoptotic action of IGF-1 in cultured cardiac myocytes

  15. Transcriptional regulation of BMP2 expression by the PTH-CREB signaling pathway in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Rongrong Zhang

    Full Text Available Intermittent application of parathyroid hormone (PTH has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP, and electrophoretic mobility shift assay (EMSA, we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.

  16. Molecular Network Analysis Suggests Aberrant CREB-Mediated Gene Regulation in the Alzheimer Disease Hippocampus

    Directory of Open Access Journals (Sweden)

    Jun-ichi Satoh

    2009-01-01

    Full Text Available The pathogenesis of Alzheimer disease (AD involves the complex interaction between genetic and environmental factors affecting multiple cellular pathways. Recent advances in systems biology provide a system-level understanding of AD by elucidating the genome-wide molecular interactions. By using KeyMolnet, a bioinformatics tool for analyzing molecular interactions on the curated knowledgebase, we characterized molecular network of 2,883 all stages of AD-related genes (ADGs and 559 incipient AD-related genes (IADGs identified by global gene expression profiling of the hippocampal CA1 region of AD brains in terms of significant clinical and pathological correlations (Blalock et al., Proc Natl Acad Sci USA 101: 2173-2178, 2004. By the common upstream search, KeyMolnet identified cAMP-response element-binding protein (CREB as the principal transcription factor exhibiting the most significant relevance to molecular networks of both ADGs and IADGs. The CREB-regulated transcriptional network included upregulated and downregulated sets of ADGs and IADGs, suggesting an involvement of generalized deregulation of the CREB signaling pathway in the pathophysiology of AD, beginning at the early stage of the disease. To verify the in silico observations in vivo, we conducted immunohistochemical studies of 11 AD and 13 age-matched control brains by using anti-phoshorylated CREB (pCREB antibody. An abnormal accumulation of pCREB imunoreactivity was identified in granules of granulovacuolar degeneration (GVD in the hippocampal neurons of AD brains. These observations suggest that aberrant CREB-mediated gene regulation serves as a molecular biomarker of AD-related pathological processes, and support the hypothesis that sequestration of pCREB in GVD granules is in part responsible for deregulation of CREB-mediated gene expression in AD hippocampus.

  17. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...

  18. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  19. Actin binding proteins and spermiogenesis

    Science.gov (United States)

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  20. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...... diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract...

  1. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

    Kragelund, B B; Knudsen, J; Poulsen, F M

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein a...

  2. CAP binding proteins associated with the nucleus.

    OpenAIRE

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in in...

  3. Insulin/IGF-I regulation of necdin and brown adipocyte differentiation via CREB- and FoxO1-associated pathways

    DEFF Research Database (Denmark)

    Cypess, Aaron M; Zhang, Hongbin; Schulz, Tim J

    2011-01-01

    , we used pharmacological inhibitors and adenoviral dominant negative constructs to demonstrate that this transition involves IRS-1 activation of Ras and ERK1/2, resulting in phosphorylation of cAMP response element-binding protein (CREB) and suppression of necdin expression. This signaling did...... not include an elevation of intracellular calcium. A constitutively active form of CREB expressed in IRS-1 knockout cells decreased necdin promoter activity, necdin mRNA, and necdin protein levels, leading to a partial restoration of differentiation. By contrast, forkhead box protein (Fox)O1, which...

  4. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  5. Inducible forebrain-specific ablation of the transcription factor Creb during adulthood induces anxiety but no spatial/contextual learning deficits

    Directory of Open Access Journals (Sweden)

    Miriam Annika Vogt

    2014-11-01

    Full Text Available The cyclic AMP (cAMP-response element binding protein (CREB is an activity-dependent transcription factor playing a role in synaptic plasticity, learning and memory, and emotional behavior. However, the impact of Creb ablation on rodent behavior is vague as e.g. memory performance of different Creb mutant mice depends on the specific type of mutation per se but additionally on the background and learning protocol differences. Here we present the first targeted ablation of CREB induced during adulthood selectively in principal forebrain neurons in a pure background strain of C57BL/6 mice. All hippocampal principal neurons exhibited lack of CREB expression. Mutant mice showed a severe anxiety phenotype in the openfield and novel object exploration test as well as in the Dark-Light Box Test, but unaltered hippocampus-dependent long-term memory in the Morris water maze and in context dependent fear conditioning. On the molecular level, CREB ablation led to CREM up regulation in the hippocampus and frontal cortex which may at least in part compensate for the loss of CREB. BDNF, a postulated CREB target gene, was down regulated in the frontal lobe but not in the hippocampus; neurogenesis remained unaltered. Our data indicate that in the adult mouse forebrain the late onset of CREB ablation can, in case of memory functionality, be compensated for and is not essential for memory consolidation and retrieval during adulthood. In contrast, the presence of CREB protein during adulthood seems to be pivotal for the regulation of emotional behavior.

  6. Characterization of a CREB Gain-of-Function Mutant with Constitutive Transcriptional Activity In Vivo

    Science.gov (United States)

    Du, Keyong; Asahara, Hiroshi; Jhala, Ulupi S.; Wagner, Brandee L.; Montminy, Marc

    2000-01-01

    The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator paralogs CREB-binding protein (CBP) and p300. CBP and p300, in turn, appear to mediate target gene induction via their association with RNA polymerase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide variety of stimuli, including hypoxia, UV irradiation, and growth factor addition, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of promoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-CBP complex formation or in the subsequent recruitment of the transcriptional apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wild-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene expression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the Km for PKA phosphorylation and thereby induces high levels of constitutive Ser133 phosphorylation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB strongly promoted differentiation of PC12 cells in concert with suboptimal doses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter. PMID:10825195

  7. Molecular Mechanisms of Stress-Induced Proenkephalin Gene Regulation: CREB Interacts with the Proenkephalin Gene in the Mouse Hypothalamus and Is Phosphorylated in Response to Hyperosmolar Stress

    Science.gov (United States)

    Borsook, David; Konradi, Christine; Falkowski, Olga; Comb, Michael; Hyman, Steven E.

    2014-01-01

    We have established a transgenic model to facilitate the study of stress-induced gene regulation in the hypothalamus. This model, which uses a human proenkephalin-β-galactosidase fusion gene, readily permits anatomic and cellular colocalization of stress-regulated immediate early gene products (e.g. Fos) and other transcription factors [e.g. cAMP response element-binding protein (CREB)] with the product of a potential target gene. Moreover, Fos provides a marker of cellular activation that is independent of the transgene. Hypertonic saline stress induced Fos in almost all cells in the PVN that exhibited basal expression of the proenkephalin transgene; however, all cells in which the transgene was activated by stress also expressed Fos. CREB was found in essentially all neurons. Gel shift analysis with and without antisera to Fos and CREB showed that AP-1 binding activity, containing Fos protein, was induced by hyperosmotic stress. However, Fos was not detected binding to the proenkephalin second messenger-inducible enhancer even in hypothalamic cell extracts from stressed animals. In contrast, CREB formed specific complexes with both the proenkephalin enhancer and a cAMP- and calcium-regulated element (CaRE) within the c-fos gene. Moreover, we found that hypertonic saline induced CREB phosphorylation in cells that express the transgene within the paraventricular nucleus and supraoptic nucleus. These results suggest a model in which proenkephalin gene expression in the paraventricular nucleus is regulated by CREB in response to hypertonic stress. PMID:8170480

  8. Moxibustion eases chronic inflammatory visceral pain through regulating MEK, ERK and CREB in rats.

    Science.gov (United States)

    Li, Zhi-Yuan; Huang, Yan; Yang, Yan-Ting; Zhang, Dan; Zhao, Yan; Hong, Jue; Liu, Jie; Wu, Li-Jie; Zhang, Cui-Hong; Wu, Huan-Gan; Zhang, Ji; Ma, Xiao-Peng

    2017-09-14

    To investigate the effects of herb-partitioned moxibustion (HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase (MEK)1, extracellular signal-regulated kinase (ERK)1/2 and cAMP response element binding protein (CREB) in spinal cord of rats with chronic inflammatory visceral pain (CIVP), and to explore the central mechanism of HPM in treating CIVP. Male Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide (DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu (ST25) and Qihai (CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor (p)MEK1, pERK1/2 and pCREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB mRNA in rat spinal cord were detected using real-time polymerase chain reaction. Compared with the normal group, the AWR scores were increased significantly ( P MEK-inhibitor groups ( P MEK-inhibitor groups. Compared with the normal group, the expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups ( P MEK, ERK and CREB mRNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups ( P MEK, ERK and CREB mRNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups ( P MEK1, ERK1/2 and CREB, and mRNA expression of MEK

  9. CREB Signaling Is Involved in Rett Syndrome Pathogenesis.

    Science.gov (United States)

    Bu, Qian; Wang, Anxin; Hamzah, Hamdi; Waldman, Alex; Jiang, Keer; Dong, Qiping; Li, Ronghui; Kim, Jason; Turner, Daniel; Chang, Qiang

    2017-03-29

    Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene. To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation ( MECP2 T158M/T158M ), hESC line expressing no MECP2 ( MECP2-KO ), congenic pair of wild-type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function. At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated from MECP2 T158M/T158M , MECP2-KO , and V247fs-MT stem cell lines. Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the female Mecp2 heterozygous mice rescued several behavioral defects. Together, our study establishes a robust in vitro platform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT. SIGNIFICANCE STATEMENT Our study establishes a robust human stem cell-based platform for consistent quantitative evaluation of genotype-dependent Rett syndrome (RTT) phenotypes at the cellular level. By providing the first evidence that enhancing cAMP response element binding protein signaling can alleviate RTT phenotypes both in vitro and in vivo , we reveal a

  10. Enhancement of behavioral sensitization, anxiety-like behavior, and hippocampal and frontal cortical CREB levels following cocaine abstinence in mice exposed to cocaine during adolescence.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Valzachi

    Full Text Available Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB and phosphorylated CREB (pCREB have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p. for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system.

  11. The effects of microwave radiation on expressions of pCREB, CREM, and CBP in the testicular tissue of rats

    Directory of Open Access Journals (Sweden)

    Hao-yu CHEN

    2012-04-01

    Full Text Available Objective To investigate the effects and ascertain the significance of microwave radiation on the expression of phosphorylated cyclic adenosine monophosphate (cAMP-response element-binding protein (pCREB, cAMP-responsive element modulator (CREM, and CREB-binding protein (CBP in the testicular tissue of rats. Methods Thirty male Wistar rats were randomly divided into control group (n=5 and radiation group (n=25. Five rats in the radiation group were sacrificed at 6h, 1, 3, 7, and 14d, respectively, after exposure to microwave radiation for 5min, with an average power density of 30mW/cm2. Rats in the control group were sacrificed within 1d without receiving microwave radiation. Expressions and dynamic changes in pCREB, CREM, and CBP in the testicular tissues were examined by immunohistochemistry and Western blotting. Results pCREB, CREM, and CBP were mainly expressed in the sperm nuclei of the seminiferous tubule in the rat testis. pCREB and CBP protein expressions were downregulated from 6h to14d (except for pCREB at 1d after exposure to microwave radiation (P < 0.05 or P < 0.01. The expression of CREM was also weakened significantly from 6h to 7d after radiation (P < 0.05 or P < 0.01. Conclusion The downregulation of pCREB, CREM, and CBP expression may play an important role in the injury of spermatogenic cells caused by microwave radiation.

  12. In Situ Protein Binding Assay Using Fc-Fusion Proteins.

    Science.gov (United States)

    Padmanabhan, Nirmala; Siddiqui, Tabrez J

    2017-01-01

    This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

  13. Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB.

    Science.gov (United States)

    Asahara, H; Santoso, B; Guzman, E; Du, K; Cole, P A; Davidson, I; Montminy, M

    2001-12-01

    The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.

  14. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  15. Long-term Administration of Salicylate-induced Changes in BDNF Expression and CREB Phosphorylation in the Auditory Cortex of Rats.

    Science.gov (United States)

    Yi, Bin; Wu, Cong; Shi, Runjie; Han, Kun; Sheng, Haibin; Li, Bei; Mei, Ling; Wang, Xueling; Huang, Zhiwu; Wu, Hao

    2018-03-01

    We investigated whether salicylate induces tinnitus through alteration of the expression levels of brain-derived neurotrophic factor (BDNF), proBDNF, tyrosine kinase receptor B (TrkB), cAMP-responsive element-binding protein (CREB), and phosphorylated CREB (p-CREB) in the auditory cortex (AC). Salicylate medication is frequently used for long-term treatment in clinical settings, but it may cause reversible tinnitus. Salicylate-induced tinnitus is associated with changes related to central auditory neuroplasticity. Our previous studies revealed enhanced neural activity and ultrastructural synaptic changes in the central auditory system after long-term salicylate administration. However, the underlying mechanisms remained unclear. Salicylate-induced tinnitus-like behavior in rats was confirmed using gap prepulse inhibition of acoustic startle and prepulse inhibition testing, followed by comparison of the expression levels of BDNF, proBDNF, TrkB, CREB, and p-CREB. Synaptic ultrastructure was observed under a transmission electron microscope. BDNF and p-CREB were upregulated along with ultrastructural changes at the synapses in the AC of rats treated chronically with salicylate (p  0.05). Long-term administration of salicylate increased BDNF expression and CREB activation, upregulated synaptic efficacy, and changed synaptic ultrastructure in the AC. There may be a relationship between these factors and the mechanism of tinnitus.

  16. Metabolic demand stimulates CREB signaling in the limbic cortex: implication for the induction of hippocampal synaptic plasticity by intrinsic stimulus for survival

    Directory of Open Access Journals (Sweden)

    Nelly M Estrada

    2009-06-01

    Full Text Available Caloric restriction by fasting has been implicated to facilitate synaptic plasticity and promote contextual learning. However, cellular and molecular mechanisms underlying the effect of fasting on memory consolidation are not completely understood. We hypothesized that fasting-induced enhancement of synaptic plasticity was mediated by the increased signaling mediated by CREB (c-AMP response element binding protein, an important nuclear protein and the transcription factor that is involved in the consolidation of memories in the hippocampus. In the in vivo rat model of 18 h fasting, the expression of phosphorylated CREB (pCREB was examined using anti-phospho-CREB (Ser133 in cardially-perfused and cryo-sectioned rat brain specimens. When compared with control animals, the hippocampus exhibited up to a two-fold of increase in pCREB expression in fasted animals. The piriform cortex, the entorhinal cortex, and the cortico-amygdala transitional zone also significantly increased immunoreactivities to pCREB. In contrast, the amygdala did not show any change in the magnitude of pCREB expression in response to fasting. The arcuate nucleus in the medial hypothalamus, which was previously reported to up-regulate CREB phosphorylation during fasting of up to 48 h, was also strongly immunoreactive and provided a positive control in the present study. Our findings demonstrate a metabolic demand not only stimulates cAMP-dependent signaling cascades in the hypothalamus, but also signals to various limbic brain regions including the hippocampus by activating the CREB signaling mechanism. The hippocampus is a primary brain structure for learning and memory. It receives hypothalamic and arcuate projections directly from the fornix. The hippocampus is also situated centrally for functional interactions with other limbic cortexes by establishing reciprocal synaptic connections. We suggest that hippocampal neurons and those in the surrounding limbic cortexes are

  17. Are many Z-DNA binding proteins actually phospholipid-binding proteins?

    OpenAIRE

    Krishna, P; Kennedy, B P; Waisman, D M; van de Sande, J H; McGhee, J D

    1990-01-01

    We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In al...

  18. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  19. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been...... described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling...

  20. Naloxone's Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptor–Gs Coupling and CREB Activation of Acute Morphine

    OpenAIRE

    Wang, Hoau-Yan; Burns, Lindsay H.

    2009-01-01

    Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Galphas and its Gbetagamma dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show t...

  1. Telomere-binding proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Zentgraf, U

    1995-02-01

    The nucleoprotein structure of Arabidopsis thaliana telomeres was investigated. A protein specifically binding to telomeric sequences was characterized by gel mobility shift assays with synthetic oligonucleotides consisting of four 7 bp telomeric repeats of Arabidopsis (TTTAGGG) and crude nuclear protein extracts of Arabidopsis leaves. These DNA-protein binding studies revealed that the binding affinity of this telomere-binding protein to the G-rich single-strand as well as to the double-stranded telomeric DNA is much higher than to the C-rich single-strand. The molecular mass of the protein was identified by SDS-PAGE to be 67 kDa. The isoelectric points were determined to be 5.0, 4.85 and 4.7, respectively, indicating that either one protein with different modifications or three slightly different proteins have been isolated. An RNA component, possibly serving as a template for reverse transcription of a plant telomerase, does not mediate the DNA-protein contact because the DNA-protein interactions were not RNAse-sensitive.

  2. Rescue of cAMP response element-binding protein signaling reversed spatial memory retention impairments induced by subanesthetic dose of propofol.

    Science.gov (United States)

    Zhang, Hao; Zhang, Shao-Bo; Zhang, Qing-Qing; Liu, Meng; He, Xing-Ying; Zou, Zui; Sun, Hai-Jing; You, Zhen-Dong; Shi, Xue-Yin

    2013-07-01

    The intravenous anesthetic propofol caused episodic memory impairments in human. We hypothesized propofol caused episodic-like spatial memory retention but not acquisition impairments in rats and rescuing cAMP response element-binding protein (CREB) signaling using selective type IV phosphodiesterase (PDEIV) inhibitor rolipram reversed these effects. Male Sprague-Dawley rats were randomized into four groups: control; propofol (25 mg/kg, intraperitoneal); rolipram; and rolipram + propofol (pretreatment of rolipram 25 min before propofol, 0.3 mg/kg, intraperitoneal). Sedation and motor coordination were evaluated 5, 15, and 25 min after propofol injection. Invisible Morris water maze (MWM) acquisition and probe test (memory retention) were performed 5 min and 24 h after propofol injection. Visible MWM training was simultaneously performed to resist nonspatial effects. Hippocampal CREB signaling was detected 5 min, 50 min, and 24 h after propofol administration. Rolipram did not change propofol-induced anesthetic/sedative states or impair motor skills. No difference was found on the latency to the platform during the visible MWM. Propofol impaired spatial memory retention but not acquisition. Rolipram reversed propofol-induced spatial memory impairments and suppression on cAMP levels, CaMKIIα and CREB phosphorylation, brain-derived neurotropic factor (BDNF) and Arc protein expression. Propofol caused spatial memory retention impairments but not acquisition inability possibly by inhibiting CREB signaling. © 2013 John Wiley & Sons Ltd.

  3. Haptenation: Chemical Reactivity and Protein Binding

    Directory of Open Access Journals (Sweden)

    Itai Chipinda

    2011-01-01

    Full Text Available Low molecular weight chemical (LMW allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

  4. Clenbuterol upregulates histone demethylase JHDM2a via the β2-adrenoceptor/cAMP/PKA/p-CREB signaling pathway.

    Science.gov (United States)

    Li, Yang; He, Jin; Sui, Shunchao; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Ning

    2012-12-01

    β(2)-Adrenergic receptor (β(2)-AR) signaling activated by the agonist clenbuterol is important in the metabolism of muscle and adipose cells. Additionally, the significant role of histone demethylase JHDM2a in regulating metabolic gene expression was also recently demonstrated in Jhdm2a(-/-) mice. To elucidate the molecular mechanism involved in clenbuterol-induced adipocyte reduction from an epigenetic perspective, this study focused on cAMP-responsive element binding protein (CREB) to determine whether JHDM2a is regulated by the β(2)-AR/cAMP/protein kinase A (PKA) signaling pathway. In porcine tissues treated with clenbuterol, JHDM2a expression was upregulated, and in porcine cells, expression of exogenous CREB led to increased JHDM2a expression. In addition, changes in JHDM2a expression were coincident with variations in the phosphorylation of CREB and p-CREB/CBP interaction in porcine and human cells treated with drugs known to modify the β(2)-AR/cAMP/PKA pathway. Finally, binding assays demonstrated that CREB regulated JHDM2a by binding directly to the CRE site nearest to the transcription start site. Our results reveal that clenbuterol activates the β(2)-AR signaling pathway upstream of JHDM2a and that CREB acts as an intermediate link regulated by cAMP-PKA to induce activity of the JHDM2a promoter. These findings suggest that clenbuterol decreases adipose cell size and increases muscle fiber size in porcine tissues by virtue of JHDM2a-mediated demethylation, which regulates downstream metabolic and related genes. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Computational search for aflatoxin binding proteins

    Science.gov (United States)

    Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

    2017-10-01

    Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

  6. Distinct changes in CREB phosphorylation in frontal cortex and striatum during contingent and non-contingent performance of a visual attention task

    Directory of Open Access Journals (Sweden)

    Mirjana eCarli

    2011-10-01

    Full Text Available The cyclic-AMP response element binding protein (CREB family of transcription factors has been implicated in numerous forms of behavioural plasticity. We investigated CREB phosphorylation along some nodes of corticostriatal circuitry such as frontal cortex (FC and dorsal (caudate putamen, CPu and ventral (nucleus accumbens, NAC striatum in response to the contingent or non-contingent performance of the five-choice serial reaction time task (5-CSRTT used to assess visuospatial attention. Three experimental manipulations were used; an attentional performance group (contingent, master, a group trained previously on the task but for whom the instrumental contingency coupling responding with stimulus detection and reward was abolished (non-contingent, yoked and a control group matched for food deprivation and exposure to the test apparatus (untrained. Rats trained on the 5-CSRTT (both master and yoked had higher levels of CREB protein in the FC, CPu and NAC compared to untrained controls. Despite the divergent behaviour of master and yoked rats CREB activity in the FC was not substantially different. In rats performing the 5-CSRTT (master, CREB activity was completely abolished in the CPu whereas in the NAC it remained unchanged. In contrast, CREB phosphorylation in CPu and NAC increased only when the contingency changed from goal-dependent to goal-independent reinforcement (yoked. The present results indicate that up-regulation of CREB protein expression across cortical and striatal regions possibly reflects the extensive instrumental learning and performance whereas increased CREB activity in striatal regions may signal the unexpected change in the relationship between instrumental action and reinforcement.

  7. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mei, E-mail: limeihit@163.com [Department of Anatomy and Neurobiology, Xuzhou Medical College, Xuzhou (China); Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing (China); Zhang, Dong-Qing; Wang, Xiang-Zhen [Department of Anatomy and Neurobiology, Xuzhou Medical College, Xuzhou (China); Xu, Tie-Jun, E-mail: xztjxu@163.com [Department of Anatomy and Neurobiology, Xuzhou Medical College, Xuzhou (China); Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing (China)

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.

  8. Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

    Science.gov (United States)

    Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling.

  9. Cyclophilin D deficiency rescues Aβ-impaired PKA/CREB signaling and alleviates synaptic degeneration.

    Science.gov (United States)

    Du, Heng; Guo, Lan; Wu, Xiaoping; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

    2014-12-01

    The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Multiple protonation equilibria in electrostatics of protein-protein binding.

    Science.gov (United States)

    Piłat, Zofia; Antosiewicz, Jan M

    2008-11-27

    All proteins contain groups capable of exchanging protons with their environment. We present here an approach, based on a rigorous thermodynamic cycle and the partition functions for energy levels characterizing protonation states of the associating proteins and their complex, to compute the electrostatic pH-dependent contribution to the free energy of protein-protein binding. The computed electrostatic binding free energies include the pH of the solution as the variable of state, mutual "polarization" of associating proteins reflected as changes in the distribution of their protonation states upon binding and fluctuations between available protonation states. The only fixed property of both proteins is the conformation; the structure of the monomers is kept in the same conformation as they have in the complex structure. As a reference, we use the electrostatic binding free energies obtained from the traditional Poisson-Boltzmann model, computed for a single macromolecular conformation fixed in a given protonation state, appropriate for given solution conditions. The new approach was tested for 12 protein-protein complexes. It is shown that explicit inclusion of protonation degrees of freedom might lead to a substantially different estimation of the electrostatic contribution to the binding free energy than that based on the traditional Poisson-Boltzmann model. This has important implications for the balancing of different contributions to the energetics of protein-protein binding and other related problems, for example, the choice of protein models for Brownian dynamics simulations of their association. Our procedure can be generalized to include conformational degrees of freedom by combining it with molecular dynamics simulations at constant pH. Unfortunately, in practice, a prohibitive factor is an enormous requirement for computer time and power. However, there may be some hope for solving this problem by combining existing constant pH molecular dynamics

  11. Up-regulation of Ca2+/CaMKII/CREB signaling in salicylate-induced tinnitus in rats.

    Science.gov (United States)

    Zhao, Jiuhan; Wang, Biao; Wang, Xiaohong; Shang, Xiuli

    2018-02-09

    The purpose of the study was to investigate the changes of Ca 2+ /calmodulin-dependent protein kinases II (CaMKII)/cAMP response element-binding protein (CREB) signaling pathway in a rat tinnitus model. Eighteen Wistar rats were randomly divided into three groups: normal control (NC), normal saline (NS), and tinnitus model (TM) groups. Tinnitus model was induced by intraperitoneal injection of salicylate. The concentration of intracellular calcium level in auditory cortex cells was determined using Fura-2 acetoxymethyl ester (Fura-2 AM) method with fluorospectrophotometer. Expressions of calmodulin (CaM), N-methyl-D-aspartate receptor 2B subunit (NR2B), calcium-calmodulin kinase II (CaMKII), and cAMP response element-binding protein (CREB) were detected with Western blot. Tinnitus model was successfully established by the intraperitoneal administration of salicylate in rats. Compared with rats in NC and NS groups, salicylate administration significantly elevated CaM, NR2B, phospho-CaMKII and phospho-CREB expression in auditory cortex from tinnitus model group (p salicylate administration causes tinnitus symptoms and elevates Ca 2+ /CaMKII/CREB signaling pathway in auditory cortex cells. Our study likely provides a new understanding of the development of tinnitus.

  12. AUXIN BINDING PROTEIN1: the outsider.

    Science.gov (United States)

    Sauer, Michael; Kleine-Vehn, Jürgen

    2011-06-01

    AUXIN BINDING PROTEIN1 (ABP1) is one of the first characterized proteins that bind auxin and has been implied as a receptor for a number of auxin responses. Early studies characterized its auxin binding properties and focused on rapid electrophysiological and cell expansion responses, while subsequent work indicated a role in cell cycle and cell division control. Very recently, ABP1 has been ascribed a role in modulating endocytic events at the plasma membrane and RHO OF PLANTS-mediated cytoskeletal rearrangements during asymmetric cell expansion. The exact molecular function of ABP1 is still unresolved, but its main activity apparently lies in influencing events at the plasma membrane. This review aims to connect the novel findings with the more classical literature on ABP1 and to point out the many open questions that still separate us from a comprehensive model of ABP1 action, almost 40 years after the first reports of its existence.

  13. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article d...

  14. A new perspective on the role of the CREB family of transcription factors in memory consolidation via adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Sylvia eOrtega-Martinez

    2015-08-01

    Full Text Available Adult neurogenesis is the process by which new neurons are generated in the brains of adults. Since its discovery 50 years ago, adult neurogenesis has been widely studied in the mammalian brain and has provided a new perspective on the pathophysiology of many psychiatric and neurodegenerative disorders, some of which affect memory. In this regard, adult hippocampal neurogenesis (AHN, which occurs in the subgranular zone of the dentate gyrus, has been suggested to play a role in the formation and consolidation of new memories. This process involves many transcription factors, of which cyclic AMP-responsive element-binding protein (CREB is a well-documented one. In the developing brain, CREB regulates crucial cell stages, (e.g., proliferation, differentiation, and survival, and in the adult brain, it participates in neuronal plasticity, learning, and memory. In addition, new evidence supports the hypothesis that CREB may also participate in learning and memory through its involvement in AHN. This review examines the CREB family of transcription factors, including the different members and known signaling pathways. It highlights the role of CREB as a modulator of AHN, which could underlie its function in memory consolidation mechanisms.

  15. A CREB1 Gene Polymorphism (rs2253206) Is Associated with Prospective Memory in a Healthy Cohort.

    Science.gov (United States)

    Avgan, Nesli; Sutherland, Heidi G; Lea, Rodney A; Spriggens, Lauren K; Haupt, Larisa M; Shum, David H K; Griffiths, Lyn R

    2017-01-01

    Prospective memory (PM) is generally defined as remembering to perform intended actions in the future and is important for functioning in daily life. Cyclic adenosine monophosphate (cAMP) responsive element binding protein 1 (CREB1) plays an important role in cognitive functions. In this study, we hypothesized that genetic variation in the CREB1 gene is associated with PM. We genotyped a CREB1 promoter polymorphism rs2253206 and tested it for association with PM in 619 healthy subjects. PM performance was measured using the Prospective and Retrospective Memory Questionnaire (PRMQ), the Comprehensive Assessment of Prospective Memory (CAPM), and the Memory for Intentions Screening Test (MIST). Generalized linear model analysis was conducted for each PM test independently using different inheritance models to identify any associations ( p CAPM instrumental activities of daily living measure ( p = 0.016). These results suggest that the rs2253206 polymorphism in the CREB1 gene locus is associated with PM in healthy individuals and contributes to knowledge on the genetics of this particular type of memory.

  16. The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila.

    Science.gov (United States)

    Wang, Biao; Goode, Jason; Best, Jennifer; Meltzer, Jodi; Schilman, Pablo E; Chen, Jian; Garza, Dan; Thomas, John B; Montminy, Marc

    2008-05-01

    In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance.

  17. A CREB1 Gene Polymorphism (rs2253206 Is Associated with Prospective Memory in a Healthy Cohort

    Directory of Open Access Journals (Sweden)

    Nesli Avgan

    2017-05-01

    Full Text Available Prospective memory (PM is generally defined as remembering to perform intended actions in the future and is important for functioning in daily life. Cyclic adenosine monophosphate (cAMP responsive element binding protein 1 (CREB1 plays an important role in cognitive functions. In this study, we hypothesized that genetic variation in the CREB1 gene is associated with PM. We genotyped a CREB1 promoter polymorphism rs2253206 and tested it for association with PM in 619 healthy subjects. PM performance was measured using the Prospective and Retrospective Memory Questionnaire (PRMQ, the Comprehensive Assessment of Prospective Memory (CAPM, and the Memory for Intentions Screening Test (MIST. Generalized linear model analysis was conducted for each PM test independently using different inheritance models to identify any associations (p < 0.05. After multiple testing adjustments, a significant association was found between the rs2253206 genotype and PM performance for CAPM instrumental activities of daily living measure (p = 0.016. These results suggest that the rs2253206 polymorphism in the CREB1 gene locus is associated with PM in healthy individuals and contributes to knowledge on the genetics of this particular type of memory.

  18. Complementarity of structure ensembles in protein-protein binding.

    Science.gov (United States)

    Grünberg, Raik; Leckner, Johan; Nilges, Michael

    2004-12-01

    Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.

  19. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

    Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

    2018-02-01

    Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (Ptannin (HT) level (Ptannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (Ptannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

  20. Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus.

    Science.gov (United States)

    Greenwood, Mingkwan; Greenwood, Michael P; Mecawi, Andre S; Loh, Su Yi; Rodrigues, José Antunes; Paton, Julian F R; Murphy, David

    2015-10-26

    Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression. The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress. Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP

  1. End binding proteins are obligatory dimers.

    Directory of Open Access Journals (Sweden)

    Indrani Sen

    Full Text Available End binding (EB proteins are responsible for the recruitment of an array of microtubule plus-end tracking proteins (+TIPs to growing microtubules ends. EBs encompass an N-terminal calponin homology domain that confers microtubule tip tracking activity to the protein. The C-terminal domain of EBs contains a coiled coil that mediates the parallel dimerization of EB monomers. This part of the protein is also responsible for partner binding. While dimerization is not essential for microtubule tip tracking by EBs it is a prerequisite for +TIP partner binding. The concentration of EBs in cells has been estimated to be in the range of hundreds of nanomoles. In contrast, in in vitro single molecule experiments EB concentrations of subnanomoles are employed. From a mechanistic point of view it is important to assess the oligomerization state of EBs at physiologically and experimentally relevant protein concentrations, in particular if the goal of a study is to model the behavior of EB-dependent dynamic +TIP networks. Here we have determined the stability of the EB1 and EB3 dimers using multi-angle light scattering and fluorescence analytical ultracentrifugation. We show that these EBs form stable dimers and do not dissociate even at very low nanomolar concentrations. The dimers remained stable at both room temperature as well as at the physiologically relevant temperature of 37°C. Together, our results reveal that EBs are obligatory dimers, a conclusion that has implications for the mechanistic understanding of these key proteins involved in the orchestration of dynamic protein networks at growing microtubule ends.

  2. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  3. CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

    Directory of Open Access Journals (Sweden)

    Ainhoa eBilbao

    2014-06-01

    Full Text Available IIt is suggested that striatal cAMP responsive element binding protein (CREB regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. Drug-naïve mutants showed moderate alterations in gene expression, most prominently a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2, when compared to wild-type controls. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB.

  4. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

    Science.gov (United States)

    Ahluwalia, Amrita; Baatar, Dolgor; Jones, Michael K; Tarnawski, Andrzej S

    2014-09-15

    Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

  5. Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

    Science.gov (United States)

    Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

    2014-04-01

    Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

  6. Binding of radionuclides to proteins in fish

    International Nuclear Information System (INIS)

    Suzuki, Yuzuru

    1981-01-01

    Radioisotope tracer experiments on binding of radionuclides to proteins in fish were carried out in order to gain further information on biochemical behavior of radionuclides in marine fish. The radionuclides, which were bound to proteins in fish through sea water and food, were extracted with a Trisacetate buffer solution and separated by gel filtration on Sephadex G-75. Most of 137 Cs in the fish liver were bound only to a peptide with a molecular weight of 1,100 - 1,300. The most remarkable feature of 60 Co in the profiles of the gel filtration was the presence of two clear radioactivity pearks and the radioactivity appeared to transfer from a low molecular weight protein to a high molecular weight protein in the case of the uptake, and the reverse phenomenon was observed in the case of the excretion. Therefore, this suggested that these proteins had each inherent turnover rate for 60 Co. The profiles of the gel filtration of 65 Zn varied widely among species of fish, tissues or organs even in the same fish and pathways of the uptake. 125 I was bound to a relatively low molecular weight substance in cultured eel, however, the binding of 125 I to a protein with higher molecular weight was observed in the eel head including thyroid gland marked through food, and this protein was estimated to be thyroglobulin with molecular weight of 670,000. Although 95 Nb, 144 Ce- 144 Pr and 106 Ru- 106 Rh probably have no biological function in fish, it was apparently found to be organically bound in tissues or organs of the marine fish. (author)

  7. Maximizing binding capacity for protein A chromatography.

    Science.gov (United States)

    Ghose, Sanchayita; Zhang, Jennifer; Conley, Lynn; Caple, Ryan; Williams, Kevin P; Cecchini, Douglas

    2014-01-01

    Advances in cell culture expression levels in the last two decades have resulted in monoclonal antibody titers of ≥10 g/L to be purified downstream. A high capacity capture step is crucial to prevent purification from being the bottleneck in the manufacturing process. Despite its high cost and other disadvantages, Protein A chromatography still remains the optimal choice for antibody capture due to the excellent selectivity provided by this step. A dual flow loading strategy was used in conjunction with a new generation high capacity Protein A resin to maximize binding capacity without significantly increasing processing time. Optimum conditions were established using a simple empirical Design of Experiment (DOE) based model and verified with a wide panel of antibodies. Dynamic binding capacities of >65 g/L could be achieved under these new conditions, significantly higher by more than one and half times the values that have been typically achieved with Protein A in the past. Furthermore, comparable process performance and product quality was demonstrated for the Protein A step at the increased loading. © 2014 American Institute of Chemical Engineers.

  8. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  9. Hepatic glucose sensing via the CREB coactivator CRTC2.

    Science.gov (United States)

    Dentin, Renaud; Hedrick, Susan; Xie, Jianxin; Yates, John; Montminy, Marc

    2008-03-07

    Chronic hyperglycemia contributes to the development of diabetes-associated complications. Increases in the concentration of circulating glucose activate the hexosamine biosynthetic pathway (HBP) and promote the O-glycosylation of proteins by O-glycosyl transferase (OGT). We show that OGT triggered hepatic gluconeogenesis through the O-glycosylation of the transducer of regulated cyclic adenosine monophosphate response element-binding protein (CREB) 2 (TORC2 or CRTC2). CRTC2 was O-glycosylated at sites that normally sequester CRTC2 in the cytoplasm through a phosphorylation-dependent mechanism. Decreasing amounts of O-glycosylated CRTC2 by expression of the deglycosylating enzyme O-GlcNAcase blocked effects of glucose on gluconeogenesis, demonstrating the importance of the HBP in the development of glucose intolerance.

  10. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...... nmol l-1 and 16 nmol l-1. The binding protein has a Stokes radius of 2.49 nm when saturated with cobalamin and 2.61 nm when unsaturated. It binds cobalamin over a broad range of pH and is able to bind cobinamide also. With immunohistochemistry, we find haptocorrin immunoreactivity in the mammary glands...

  11. Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model

    Directory of Open Access Journals (Sweden)

    Xiao-Mei Shao

    2016-01-01

    Full Text Available Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs in a rat pain memory model. To explore the critical role of protein kinase A (PKA in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP, PKA, cAMP response element-binding protein (CREB, and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

  12. Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model.

    Science.gov (United States)

    Shao, Xiao-Mei; Sun, Jing; Jiang, Yong-Liang; Liu, Bo-Yi; Shen, Zui; Fang, Fang; Du, Jun-Ying; Wu, Yuan-Yuan; Wang, Jia-Ling; Fang, Jian-Qiao

    2016-01-01

    Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

  13. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  14. Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

    Science.gov (United States)

    Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik; Fahrenkrug, Jan; Stehle, Jörg H

    2016-09-01

    Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus

  15. What Happened to the IGF Binding Proteins?

    Science.gov (United States)

    Bach, Leon A

    2018-02-01

    Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

  16. Cross-Sectional and Longitudinal Effects of CREB1 Genotypes on Individual Differences in Memory and Executive Function: Findings from the BLSA

    Directory of Open Access Journals (Sweden)

    Claudia Wolf

    2017-05-01

    Full Text Available Purpose: Previously, we have shown that the SNP rs10932201 genotype of the cyclic AMP responsive element binding protein 1 gene (CREB1 contributes to individual differences in executive and memory function at the neural system and behavioral levels in healthy, young adults. However, longitudinal effects of CREB1 genotypes on cognition have not yet been addressed. Furthermore we were interested in replicating associations between CREB1 genotypes and human cognition in previous cross-sectional studies and explore whether APOE4 status might modify these relations.Materials and Methods: We investigated whether common, independent tag SNPs within CREB1 (rs2253206, rs10932201, rs6785 influence individual differences in age-related longitudinal change and level of executive function and memory performance independent of baseline age, sex, APOE4 status, and education. Our analysis included data from cognitively unimpaired older adults participating in the Baltimore Longitudinal Study of Aging. Eleven measures from six cognitive tests (sample sizes range 617–786 were analyzed using linear mixed effects and generalized estimating equations models. Mean baseline age ranged from 50 to 69 years and mean time of follow-up (interval ranged from 8 to 22 years.Results: We found significant effects of all three CREB1 SNPs on performance level and/or longitudinal change in performance based on eight measures assessing semantic memory, episodic memory, or both executive function and semantic memory. SNP rs10932201 showed the most significant and largest effect (Cohen’s d = -0.70, p < 0.01 on age-related longitudinal decline of semantic memory. Additionally, we show interactions between all three CREB1 SNPs and APOE4 status on age-related longitudinal declines and levels of memory and executive function.Conclusion: Our results suggest that CREB1 genotypes independently and by interactions with APOE4 status contribute to individual differences in cognitive aging.

  17. The CREB Coactivator CRTC2 Is a Lymphoma Tumor Suppressor that Preserves Genome Integrity through Transcription of DNA Mismatch Repair Genes.

    Science.gov (United States)

    Fang, Minggang; Pak, Magnolia L; Chamberlain, Lynn; Xing, Wei; Yu, Hongbo; Green, Michael R

    2015-06-09

    The CREB-regulated transcription coactivator CRTC2 stimulates CREB target gene expression and has a well-established role in modulating glucose and lipid metabolism. Here, we find, unexpectedly, that loss of CRTC2, as well as CREB1 and its coactivator CREB-binding protein (CBP), results in a deficiency in DNA mismatch repair (MMR) and a resultant increased mutation frequency. We show that CRTC2, CREB1, and CBP are transcriptional activators of well-established MMR genes, including EXO1, MSH6, PMS1, and POLD2. Mining of expression profiling databases and analysis of patient samples reveal that CRTC2 and its target MMR genes are downregulated in specific T cell lymphoma subtypes, which are microsatellite unstable. The levels of acetylated histone H3 on the CRTC2 promoter are significantly reduced in lymphoma in comparison to normal tissue, explaining the decreased CRTC2 expression. Our results establish a role for CRTC2 as a lymphoma tumor suppressor gene that preserves genome integrity by stimulating transcription of MMR genes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  19. Ice-Binding Proteins in Plants

    Directory of Open Access Journals (Sweden)

    Melissa Bredow

    2017-12-01

    Full Text Available Sub-zero temperatures put plants at risk of damage associated with the formation of ice crystals in the apoplast. Some freeze-tolerant plants mitigate this risk by expressing ice-binding proteins (IBPs, that adsorb to ice crystals and modify their growth. IBPs are found across several biological kingdoms, with their ice-binding activity and function uniquely suited to the lifestyle they have evolved to protect, be it in fishes, insects or plants. While IBPs from freeze-avoidant species significantly depress the freezing point, plant IBPs typically have a reduced ability to lower the freezing temperature. Nevertheless, they have a superior ability to inhibit the recrystallization of formed ice. This latter activity prevents ice crystals from growing larger at temperatures close to melting. Attempts to engineer frost-hardy plants by the controlled transfer of IBPs from freeze-avoiding fish and insects have been largely unsuccessful. In contrast, the expression of recombinant IBP sequences from freeze-tolerant plants significantly reduced electrolyte leakage and enhanced freezing survival in freeze-sensitive plants. These promising results have spurred additional investigations into plant IBP localization and post-translational modifications, as well as a re-evaluation of IBPs as part of the anti-stress and anti-pathogen axis of freeze-tolerant plants. Here we present an overview of plant freezing stress and adaptation mechanisms and discuss the potential utility of IBPs for the generation of freeze-tolerant crops.

  20. Apolipoprotein B is a calcium binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Dashti, N.; Lee, D.M.; Mok, T.

    1986-05-29

    Human hepatocarcinoma Hep G2 cells were grown in culture medium containing (/sup 45/Ca/sup 2 +/). The secreted lipoproteins of d < 1.063 g/ml and d 1.063-1.21 g/ml were isolated from the culture media and analyzed by 3.3% and 7% SDS-polyacrylamide gel electrophoresis. Radioactivity profiles of (/sup 45/Ca) from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated (/sup 45/Ca/sup 2 +/) and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein.

  1. Pharmacological Activators of the NR4A Nuclear Receptors Enhance LTP in a CREB/CBP-Dependent Manner.

    Science.gov (United States)

    Bridi, Morgan S; Hawk, Joshua D; Chatterjee, Snehajyoti; Safe, Stephen; Abel, Ted

    2017-05-01

    Nr4a nuclear receptors contribute to long-term memory formation and are required for long-term memory enhancement by a class of broad-acting drugs known as histone deacetylase (HDAC) inhibitors. Understanding the molecular mechanisms that regulate these genes and identifying ways to increase their activity may provide novel therapeutic approaches for ameliorating cognitive dysfunction. In the present study, we find that Nr4a gene expression after learning requires the cAMP-response element binding (CREB) interaction domain of the histone acetyltransferase CREB-binding protein (CBP). These gene expression deficits emerge at a time after learning marked by promoter histone acetylation in wild-type mice. Further, mutation of the CREB-CBP interaction domain reduces Nr4a promoter acetylation after learning. As memory enhancement by HDAC inhibitors requires CREB-CBP interaction and Nr4a gene function, these data support the notion that the balance of histone acetylation at the Nr4a promoters is critical for memory formation. NR4A ligands have recently been described, but the effect of these drugs on synaptic plasticity or memory has not been investigated. We find that the 'C-DIM' NR4A ligands, para-phenyl substituted di-indolylmethane compounds, enhance long-term contextual fear memory and increase the duration of long-term potentiation (LTP), a form of hippocampal synaptic plasticity. LTP enhancement by these drugs is eliminated in mice expressing a dominant negative form of NR4A and attenuated in mice with mutation of the CREB-CBP interaction domain. These data define the molecular connection between histone acetylation and Nr4a gene expression after learning. In addition, they suggest that NR4A-activating C-DIM compounds may serve as a potent and selective means to enhance memory and synaptic plasticity.

  2. Glutamatergic induction of CREB phosphorylation and Fos expression in primary cultures of the suprachiasmatic hypothalamus in vitro is mediated by co-ordinate activity of NMDA and non-NMDA receptors.

    Science.gov (United States)

    Schurov, I L; McNulty, S; Best, J D; Sloper, P J; Hastings, M H

    1999-01-01

    Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induced nuclear immunoreactivity (-ir) to the phospho-Ser133 form of the Ca2+/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear Fos-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl-D-aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB-ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino-methyl proprionic acid (AMPA), and Kainate (KA)), whereas total CREB did not change. Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate caused a rapid (15 min) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurones, both GABA-ir and others. Treatment with NMDA alone had no effect on pCREB-ir. AMPA alone caused a slight increase in pCREB-ir. However, kainate alone or in combination with NMDA and AMPA induced nuclear pCREB-ir equal to that induced by glutamate. The effects of glutamate on pCREB-ir and Fos-ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg2+, MK801 blocked glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist was no longer effective at blocking glutamatergic induction of either Fos-ir or pCREB-ir, consistent with the model that glutamate regulates gene expression in the SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB-ir in GABA-ir neurones was blocked by KN-62 an inhibitor of Ca2+/Calmodulin (Ca

  3. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  4. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  5. In vitro binding of germanium to proteins of rice shoots

    International Nuclear Information System (INIS)

    Matsumoto, Hideaki; Takahashi, Eiichi

    1976-01-01

    The possibility of in vitro binding between proteins of rice shoots and germanium (Ge) was investigated. The proteins in mixtures of aqueous extracts of rice shoots and radioactive germanium ( 68 GeO 2 ) were fractionated. The binding of radioactivity to the proteins was observed even after 5 successive fractionation steps from the original mixtures. At the final fractionation step using polyacrylamide gel electrophoresis, a constant proportionality between protein concentration and associated radioactivity was found in most samples although not all. These results indicate that the binding of 68 Ge to proteins is not due to the simple adsorption by proteins. (auth.)

  6. Development of radioimmunoassay for prolactin binding protein

    International Nuclear Information System (INIS)

    Raikar, R.S.; Sheth, A.R.

    1982-01-01

    Using a homogenous prolactin binding protein (PBP) preparations from rat seminal vesicle secretion, a sensitive and specific radioimmunoassay (RIA) for PBP has been developed. The assay was highly specific and showed no cross-reaction with other protein hormones from various species. The antiserum had an affinity constant (Ka) of 2.66 x 10 10 M -1 . The assay sensitivity was in the range of 0.5-1.0 ng of pure PBP per assay tube and the intra- and inter-assay coefficients of variations were 6-8% and 12-14.5% respectively. The overall recovery of PBP to the rat seminal vesicle secretion was 96.8%. Using this RIA, PBP levels in various biological fluids and reproductive tissues were measured. Azoospermic human semen contained significantly higher levels of PBP than normospermic semen. The seminal vesicle of rat exhibited the highest concentration of PBP. Administration of antiserum to PBP to mature male rats resulted in a significant reduction in the weight of ventral prostrate and serum prolactin levels were significantly elevated in these animals suggesting that the antibody raised against the PBP was capable of blocking prolactin receptors. (author)

  7. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  8. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  9. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  10. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  11. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-01-01

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

  12. Diethylstilbestrol regulates mouse gubernaculum testis cell proliferation via PLC-Ca2+-CREB pathway.

    Science.gov (United States)

    Zhang, Xuan; Ping, Hong-Yan; Li, Jian-Hong; Duan, Shou-Xin; Jiang, Xue-Wu

    2018-01-01

    Recent evidence suggested a positive correlation between environmental estrogens (EEs) and high incidence of abnormalities in male urogenital system, but the mechanism remains unclear. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernaculum testis cells, but the underlying mechanism is unclear. In this study, mouse gubernaculum testis cells were pretreated with phospholipase C (PLC) inhibitor U-73122 and then treated with DES. The results demonstrated that U-73122 impaired DES-evoked intracellular Ca2+ mobilization in gubernaculum testis cells and inhibited DES-induced proliferation of gubernaculum testis cells. Mechanistically, we found that U-73122 inhibited DES-induced activation of cAMP-response element binding protein (CREB) in gubernaculum testis cells. In conclusion, these data suggest that the effects of DES on mouse gubernaculum testis cells are mediated by PLC-Ca 2+ -CREB pathway. Environmental estrogens remain a serious threat to male reproductive health, and it is important to understand the mechanism by which EEs affect the male productive system. Here we explore potential mechanisms how the proliferation and contractility of gubernaculum testis cells are regulated by diethylstilbestrol. Our findings provide the first evidence that PLC-Ca 2+ -CREB signalling pathway mediates the nongenomic effects of diethylstilbestrol on gubernaculum testis cells. These findings provide new insight into the role of diethylstilbestrol in the aetiology of male reproductive dysfunction and will help develop better approaches for the prevention and therapy of male reproductive malformation. Copyright © 2017 John Wiley & Sons, Ltd.

  13. DPPC regulates COX-2 expression in monocytes via phosphorylation of CREB

    International Nuclear Information System (INIS)

    Morris, R.H.K.; Tonks, A.J.; Jones, K.P.; Ahluwalia, M.K.; Thomas, A.W.; Tonks, A.; Jackson, S.K.

    2008-01-01

    The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE 2 (P < 0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-κB activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using L-α-phosphatidylcholine β-arachidonoyl-γ-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2

  14. CREB-mediated alterations in the amygdala transcriptome: coordinated regulation of immune response genes following cocaine.

    Science.gov (United States)

    Ecke, Laurel E; Cleck, Jessica N; White, Peter; Schug, Jonathan; Mifflin, Lauren; Blendy, Julie A

    2011-09-01

    The neuronal circuitry underlying stress- and drug-induced reinstatement of cocaine-seeking has been relatively well characterized; however, less is known regarding the long-term molecular changes following cocaine administration that may promote future reinstatement. The transcription factor cAMP response element-binding protein (CREB) is necessary for stress- but not cocaine-induced reinstatement of conditioned reward, suggesting that different molecular mechanisms may underlie these two types of reinstatement. To explore the relationship between this transcription factor and reinstatement, we utilized the place-conditioning paradigm to examine alterations in gene expression in the amygdala, a neural substrate critically involved in stress-induced reinstatement, following the development of cocaine reward and subsequent extinction. Our findings demonstrate that the amygdala transcriptome was altered by CREB deficiency more than by previous cocaine experience, with an over-representation of genes involved in the immune response. However, a subset of genes involved in stress and immune response demonstrated a drug×genotype interaction, indicating that cocaine produces different long-term alterations in gene expression depending on the presence or absence of CREB. This profile of gene expression in the context of addiction enhances our understanding of the long-term molecular changes that occur throughout the addiction cycle and identifies novel genes and pathways that might lead to the creation of better therapeutic agents.

  15. The Creb1 coactivator Crtc1 is required for energy balance and fertility.

    Science.gov (United States)

    Altarejos, Judith Y; Goebel, Naomi; Conkright, Michael D; Inoue, Hiroshi; Xie, Jianxin; Arias, Carlos M; Sawchenko, Paul E; Montminy, Marc

    2008-10-01

    The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (Leprs) that act on the signal transducer and activator of transcription 3 (Stat3). Although disruption of Lepr-Stat3 signaling promotes obesity in mice, other features of Lepr function, such as fertility, seem normal, pointing to the involvement of additional regulators. Here we show that the cyclic AMP responsive element-binding protein-1 (Creb1)-regulated transcription coactivator-1 (Crtc1) is required for energy balance and reproduction-Crtc1(-/-) mice are hyperphagic, obese and infertile. Hypothalamic Crtc1 was phosphorylated and inactive in leptin-deficient ob/ob mice, while leptin administration increased amounts of dephosphorylated nuclear Crtc1. Dephosphorylated Crtc1 stimulated expression of the Cartpt and Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptin's effects on satiety and fertility. Crtc1 overexpression in hypothalamic cells increased Cartpt and Kiss1 gene expression, whereas Crtc1 depletion decreased it. Indeed, leptin enhanced Crtc1 activity over the Cartpt and Kiss1 promoters in cells overexpressing Lepr, and these effects were disrupted by expression of a dominant-negative Creb1 polypeptide. As leptin administration increased recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters, our results indicate that the Creb1-Crtc1 pathway mediates the central effects of hormones and nutrients on energy balance and fertility.

  16. The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.

    Science.gov (United States)

    Koo, Seung-Hoi; Flechner, Lawrence; Qi, Ling; Zhang, Xinmin; Screaton, Robert A; Jeffries, Shawn; Hedrick, Susan; Xu, Wu; Boussouar, Fayçal; Brindle, Paul; Takemori, Hiroshi; Montminy, Marc

    2005-10-20

    Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon, and at the cellular level by energy status. Glucagon enhances glucose output from the liver during fasting by stimulating the transcription of gluconeogenic genes via the cyclic AMP-inducible factor CREB (CRE binding protein). When cellular ATP levels are low, however, the energy-sensing kinase AMPK inhibits hepatic gluconeogenesis through an unknown mechanism. Here we show that hormonal and energy-sensing pathways converge on the coactivator TORC2 (transducer of regulated CREB activity 2) to modulate glucose output. Sequestered in the cytoplasm under feeding conditions, TORC2 is dephosphorylated and transported to the nucleus where it enhances CREB-dependent transcription in response to fasting stimuli. Conversely, signals that activate AMPK attenuate the gluconeogenic programme by promoting TORC2 phosphorylation and blocking its nuclear accumulation. Individuals with type 2 diabetes often exhibit fasting hyperglycaemia due to elevated gluconeogenesis; compounds that enhance TORC2 phosphorylation may offer therapeutic benefits in this setting.

  17. Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity.

    Science.gov (United States)

    Wang, Yiguo; Inoue, Hiroshi; Ravnskjaer, Kim; Viste, Kristin; Miller, Nina; Liu, Yi; Hedrick, Susan; Vera, Liliana; Montminy, Marc

    2010-02-16

    Under fasting conditions, increases in circulating concentrations of pancreatic glucagon maintain glucose homeostasis through induction of gluconeogenic genes by the CREB coactivator CRTC2. Hepatic CRTC2 activity is elevated in obesity, although the extent to which this cofactor contributes to attendant increases in insulin resistance is unclear. Here we show that mice with a knockout of the CRTC2 gene have decreased circulating glucose concentrations during fasting, due to attenuation of the gluconeogenic program. CRTC2 was found to stimulate hepatic gene expression in part through an N-terminal CREB binding domain that enhanced CREB occupancy over relevant promoters in response to glucagon. Deletion of sequences encoding the CREB binding domain in CRTC2 (-/-) mice lowered circulating blood glucose concentrations and improved insulin sensitivity in the context of diet-induced obesity. Our results suggest that small molecules that attenuate the CREB-CRTC2 pathway may provide therapeutic benefit to individuals with type 2 diabetes.

  18. Thermodynamics of Ligand Binding to Acyl-Coenzyme A Binding Protein Studied by Titration Calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils Joakim; Sigurskjold, Bent Walther; Kragelund, Birthe B.

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  19. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  20. SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

    Science.gov (United States)

    Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

    2016-10-20

    RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

    DEFF Research Database (Denmark)

    Jessen, U; Novitskaya, V; Pedersen, N

    2001-01-01

    roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... form of the MAPK kinase, MEK2. Alternatively, PC12-E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c-Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c......The neural cell adhesion molecule (NCAM) stimulates axonal outgrowth by activation of the Ras-mitogen activated protein kinase (MAPK) pathway and by generation of arachidonic acid. We investigated whether the transcription factors, cyclic-AMP response-element binding protein (CREB) and c-Fos play...

  2. Actin binding proteins, spermatid transport and spermiation*

    Science.gov (United States)

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  3. Interleukin-18 and interleukin-18 Binding Protein

    Directory of Open Access Journals (Sweden)

    Charles eDinarello

    2013-10-01

    Full Text Available Interleukin-18 (IL 18 is a member of the IL 1 family of cytokines. Increasing reports have expanded the role of IL 18 in mediating inflammation in animal models of disease using IL 18 deficient mice, neutralization of IL 18 or deficiency in the IL 18 receptor alpha chain. Similar to IL 1β, IL 18 is synthesized as an inactive precursor requiering processing by caspase 1 into an active cytokine but unlike IL 1β, the IL 18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL 18 is balanced by the presence of a high-affinity naturally occuring IL 18 binding protein (IL 18BP. In humans, disease increased disease severity can be associated with an imbalance of IL 18 to IL 18BP such that the levels of free IL 18 are elevated in the circulation. A role for IL 18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis and acute kidney injury, although in some diseases, IL 18 is protective. IL 18 plays a major role in the production of interferon-g from natural killer cells. The IL 18BP has been used safely in humans and clinical trials of IL 18BP as well as neutralizing anti-IL 18 antibodies are in clinical trials. This review updates the biology of IL 18 as well as its role in human disease

  4. IGF Binding Protein-5 Induces Cell Senescence

    Directory of Open Access Journals (Sweden)

    Fumihiro Sanada

    2018-02-01

    Full Text Available Cellular senescence is the complex process of deterioration that drives the aging of an organism, resulting in the progressive loss of organ function and eventually phenotypic aging. Senescent cells undergo irreversible growth arrest, usually by inducing telomere shortening. Alternatively, senescence may also occur prematurely in response to various stress stimuli, such as oxidative stress, DNA damage, or activated oncogenes. Recently, it has been shown that IGF binding protein-5 (IGFBP-5 with the induction of the tumor suppressor p53 is upregulated during cellular senescence. This mechanism mediates interleukin-6/gp130-induced premature senescence in human fibroblasts, irradiation-induced premature senescence in human endothelial cells (ECs, and replicative senescence in human ECs independent of insulin-like growth factor I (IGF-I and IGF-II. Additionally, a link between IGFBP-5, hyper-coagulation, and inflammation, which occur with age, has been implicated. Thus, IGFBP-5 seems to play decisive roles in controlling cell senescence and cell inflammation. In this review, we describe the accumulating evidence for this role of IGFBP-5 including our new finding.

  5. Immobilized purified folate-binding protein: binding characteristics and use for quantifying folate in erythrocytes

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Nexo, E.

    1987-01-01

    Purified folate-binding protein from cow's milk was immobilized on monodisperse polymer particles (Dynospheres) activated by rho-toluenesulfonyl chloride. Leakage from the spheres was less than 0.1%, and the binding properties were similar to those of the soluble protein with regard to dissociation, pH optimum for binding pteroylglutamic acid, and specificity for binding various folate derivatives. We used the immobilized folate-binding protein as binding protein in an isotope-dilution assay for quantifying folate in erythrocytes. The detection limit was 50 nmol/L and the CV over a six-month period was 2.3% (means = 1.25 mumol/L, n = 15). The reference interval, for folate measured in erythrocytes of 43 blood donors, was 0.4-1.5 mumol/L

  6. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  7. SCOWLP classification: Structural comparison and analysis of protein binding regions

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

    Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

  8. Accurate prediction of peptide binding sites on protein surfaces.

    Directory of Open Access Journals (Sweden)

    Evangelia Petsalaki

    2009-03-01

    Full Text Available Many important protein-protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled but where no structure of the protein-peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein-peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.

  9. Antigenic and structural conservation of herpesvirus DNA-binding proteins.

    Science.gov (United States)

    Littler, E; Yeo, J; Killington, R A; Purifoy, D J; Powell, K L

    1981-10-01

    Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpes viruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.

  10. Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

    Science.gov (United States)

    Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

    2016-11-01

    Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

  11. A brave new world of RNA-binding proteins.

    Science.gov (United States)

    Hentze, Matthias W; Castello, Alfredo; Schwarzl, Thomas; Preiss, Thomas

    2018-01-17

    RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple globular RNA-binding domains (RBDs) and change the fate or function of the bound RNAs. Several hundred such RBPs have been discovered and investigated over the years. Recent proteome-wide studies have more than doubled the number of proteins implicated in RNA binding and uncovered hundreds of additional RBPs lacking conventional RBDs. In this Review, we discuss these new RBPs and the emerging understanding of their unexpected modes of RNA binding, which can be mediated by intrinsically disordered regions, protein-protein interaction interfaces and enzymatic cores, among others. We also discuss the RNA targets and molecular and cellular functions of the new RBPs, as well as the possibility that some RBPs may be regulated by RNA rather than regulate RNA.

  12. Short term memory of Caenorhabditis elegans against bacterial pathogens involves CREB transcription factor.

    Science.gov (United States)

    Prithika, Udayakumar; Vikneswari, Ramaraj; Balamurugan, Krishnaswamy

    2017-04-01

    One of the key issues pertaining to the control of memory is to respond to a consistently changing environment or microbial niche present in it. Human cyclic AMP response element binding protein (CREB) transcription factor which plays a crucial role in memory has a homolog in C. elegans, crh-1. crh-1 appears to influence memory processes to certain extent by habituation of the host to a particular environment. The discrimination between the pathogen and a non-pathogen is essential for C. elegans in a microbial niche which determines its survival. Training the nematodes in the presence of a virulent pathogen (S. aureus) and an opportunistic pathogen (P. mirabilis) separately exhibits a different behavioural paradigm. This appears to be dependent on the CREB transcription factor. Here we show that C. elegans homolog crh-1 helps in memory response for a short term against the interacting pathogens. Following conditioning of the nematodes to S. aureus and P. mirabilis, the wild type nematodes exhibited a positive response towards the respective pathogens which diminished slowly after 2h. By contrast, the crh-1 deficient nematodes had a defective memory post conditioning. The molecular data reinforces the importance of crh-1 gene in retaining the memory of nematode. Our results also suggest that involvement of neurotransmitters play a crucial role in modulating the memory of the nematode with the assistance of CREB. Therefore, we elucidate that CREB is responsible for the short term memory response in C. elegans against bacterial pathogens. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. Morphine Reward Promotes Cue-Sensitive Learning: Implication of Dorsal Striatal CREB Activity

    Directory of Open Access Journals (Sweden)

    Mathieu Baudonnat

    2017-05-01

    Full Text Available Different parallel neural circuits interact and may even compete to process and store information: whereas stimulus–response (S–R learning critically depends on the dorsal striatum (DS, spatial memory relies on the hippocampus (HPC. Strikingly, despite its potential importance for our understanding of addictive behaviors, the impact of drug rewards on memory systems dynamics has not been extensively studied. Here, we assessed long-term effects of drug- vs food reinforcement on the subsequent use of S–R vs spatial learning strategies and their neural substrates. Mice were trained in a Y-maze cue-guided task, during which either food or morphine injections into the ventral tegmental area (VTA were used as rewards. Although drug- and food-reinforced mice learned the Y-maze task equally well, drug-reinforced mice exhibited a preferential use of an S–R learning strategy when tested in a water-maze competition task designed to dissociate cue-based and spatial learning. This cognitive bias was associated with a persistent increase in the phosphorylated form of cAMP response element-binding protein phosphorylation (pCREB within the DS, and a decrease of pCREB expression in the HPC. Pharmacological inhibition of striatal PKA pathway in drug-rewarded mice limited the morphine-induced increase in levels of pCREB in DS and restored a balanced use of spatial vs cue-based learning. Our findings suggest that drug (opiate reward biases the engagement of separate memory systems toward a predominant use of the cue-dependent system via an increase in learning-related striatal pCREB activity. Persistent functional imbalance between striatal and hippocampal activity could contribute to the persistence of addictive behaviors, or counteract the efficiency of pharmacological or psychotherapeutic treatments.

  14. Identification of calcium-binding proteins in human platelets

    International Nuclear Information System (INIS)

    Brumley, L.M.; Wallace, R.W.

    1986-01-01

    In human platelets, intracellular Ca 2+ is a second messenger for platelet agonists. Two targets for the Ca 2+ signal are calmodulin and the C-,inase; however, other Ca 2+ -binding proteins may also play a role in platelet function. The Western blotting technique of Maruyama et al., which utilizes 45 Ca 2+ to detect Ca 2+ -binding proteins, has been used to identify numerous platelet Ca 2+ -binding proteins ranging in molecular weight from 165K to 15K. The greatest quantity of 45 Ca 2+ was bound to a 165 kilodalton protein which has been identified as thrombospondin based upon its release from thrombin-stimulated platelets and its comigration on SDS gels with purified thrombospondin. Two other major sites for 45 Ca 2+ -binding correspond to proteins of 120K and 108K which are present only in the platelet particulate fraction; they have been identified as glycoproteins IIb and IIIa based upon their labeling by 125 I-concanavalin A. Two proteins with molecular weights of 20K and 15K bound much less 45 Ca 2+ and correspond on SDS gels to calmodulin and subunit B of the calmodulin-dependent phosphatase. A number of other, yet to be identified, Ca 2+ -binding proteins were also detected. These data indicate that human platelets contain numerous Ca 2+ -binding proteins and that Western blotting techniques utilizing 45 Ca 2+ may be useful as an assay system in future attempts to purify platelet Ca 2+ -binding proteins

  15. Rate Constants and Mechanisms of Protein-Ligand Binding.

    Science.gov (United States)

    Pang, Xiaodong; Zhou, Huan-Xiang

    2017-05-22

    Whereas protein-ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms.

  16. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current...

  17. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  18. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  19. Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Zhong-Rong Zhang

    2017-06-01

    Full Text Available Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L. Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB. Blockage of cAMP/CREB downstream signals with protein kinase A (PKA inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

  20. Epigenetic modification of miR-10a regulates renal damage by targeting CREB1 in type 2 diabetes mellitus

    International Nuclear Information System (INIS)

    Shan, Qun; Zheng, Guihong; Zhu, Aihua; Cao, Li; Lu, Jun; Wu, Dongmei; Zhang, ZiFeng; Fan, Shaohua; Sun, Chunhui; Hu, Bin; Zheng, Yuanlin

    2016-01-01

    Emerging evidence has shown that microRNA-mediated gene expression modulation plays a crucial role in the pathogenesis of type 2 diabetes mellitus, but the novel miRNAs involved in type 2 diabetes and its functional regulatory mechanisms still need to be determined. In this study, we assessed the role of miR-10a in extracellular matrix accumulation in the kidney of diabetic mellitus induced by combining administration of chronic high fat diet (HFD) and low dosage of streptozotocin (STZ, 35 mg/kg). Here, we found that HFD/STZ administration decreased the level of microRNA (miR-10a) expression in ICR strain mice. Overexpression of miR-10a alleviated the increased ratio of urine albumin-to-creatinine (ACR) ratio of HFD/STZ mice. In contrast, knockdown of miR-10a increased the ratio of kidney ACR in naïve mice. Furthermore, cAMP response element binding protein 1 (CREB1) was validated as a target of miR-10a in vitro and in vivo. CREB1 and its downstream fibronectin (FN, extracellular matrix) were increased in HFD/STZ-treated mice, which was reversed by kidney miR-10a overexpression. The content of CREB1 and FN was increased by miR-10a knockdown in kidney of naïve mice. Furthermore, histone deacetylase 3 (HDAC3) was revealed to be increased in kidney of HFD/STZ mice, accompanied with the augmentation of ACR ratio and FN level. Knockdown of HDAC3 with siRNA significantly caused the increase of miR-10a, resulting in the decrease in CREB1 and FN expression in kidney of HFD/STZ mice. Contrarily, HDAC3 overexpression mediated by lentivirus decreased miR-10a content, and enhanced ACR value, CREB1 and FN formation in naïve mice. Collectively, these results elucidate that HDAC3/miR-10a/CREB1 serves as a new mechanism underlying kidney injury, providing potential therapeutic targets in type 2 diabetes. - Highlights: • Diabetes induces the decrease of miR-10a level in the kidney. • MiR-10a overexpression improves kidney damage of diabetes. • MiR-10a targeting CREB1/FN

  1. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  2. Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

    Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

  3. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

    International Nuclear Information System (INIS)

    Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

    2015-01-01

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

  4. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Wei-Jie; Wang, Sheng [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Hu, Zhuang, E-mail: zhuanghu475000@sina.com [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China); Zhou, Zhen-Yu; Song, Cai-Juan [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China)

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

  5. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  6. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  7. Discrete persistent-chain model for protein binding on DNA.

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2011-04-01

    We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

  8. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  9. Predicting nucleic acid binding interfaces from structural models of proteins.

    Science.gov (United States)

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2012-02-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

  10. New binding mode to TNF-alpha revealed by ubiquitin-based artificial binding protein.

    Directory of Open Access Journals (Sweden)

    Andreas Hoffmann

    Full Text Available A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins--designed ankyrin repeat proteins--without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

  11. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  12. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan (China); Tang, Ming-Chi [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Kuo, Liang-Mou [Department of General Surgery, Chang Gung Memorial Hospital at Chia-Yi, Taiwan (China); Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China)

    2012-04-15

    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  13. Dorsal hippocampal NMDA receptor blockade impairs extinction of naloxone-precipitated conditioned place aversion in acute morphine-treated rats by suppressing ERK and CREB phosphorylation in the basolateral amygdala.

    Science.gov (United States)

    Wang, Wei-Sheng; Chen, Zhong-Guo; Liu, Wen-Tao; Chi, Zhi-Qiang; He, Ling; Liu, Jing-Gen

    2015-01-01

    Substantial evidence shows that negative reinforcement resulting from the aversive affective consequences of opiate withdrawal may play a crucial role in drug relapse. Understanding the mechanisms underlying the loss (extinction) of conditioned aversion of drug withdrawal could facilitate the treatment of drug addiction. Naloxone-induced conditioned place aversion (CPA) of Sprague-Dawley rats was used to measure conditioned aversion. An NMDA receptor antagonist and MAPK kinase inhibitor were applied through intracranial injections. The phosphorylation of ERK and cAMP response element-binding protein (CREB) was detected using Western blot. The extinction of CPA behaviour increased the phosphorylation of ERK and CREB in the dorsal hippocampus (DH) and basolateral amygdala (BLA), but not in the central amygdala (CeA). Intra-DH injection of AP5 or intra-BLA injection of AP-5 or U0126 before extinction training significantly attenuated ERK and CREB phosphorylation in the BLA and impaired the extinction of CPA behaviour. Although intra-DH injections of AP-5 attenuated extinction training-induced activation of the ERK-CREB pathway in the BLA, intra-BLA injection of AP5 had no effect on extinction training-induced activation of the ERK-CREB pathway in the DH. These results suggest that activation of ERK and CREB in the BLA and DH is involved in the extinction of CPA behaviour and that the DH, via a direct or indirect pathway, modulates the activity of ERK and CREB in the BLA through activation of NMDA receptors after extinction training. Understanding the mechanisms underlying the extinction of conditioned aversion could facilitate the treatment of drug addiction. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  14. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of ...

  15. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple

  16. Measurement of plasma protein and lipoprotein binding of pyrethroids.

    Science.gov (United States)

    Sethi, Pankaj K; Muralidhara, S; Bruckner, James V; White, Catherine A

    2014-01-01

    A simple, reliable procedure was developed to measure binding of pyrethroid insecticides to total proteins and lipoproteins of rat and human plasma. The extent of binding of (14)C-labeled deltamethrin (DLM), cis-permethrin (CIS) and trans-permethrin (TRANS) was quantified by a 3-step organic solvent extraction technique. Rat and human plasma samples, containing NaF to inhibit esterases, were spiked with a range of concentrations of each radiolabeled pyrethroid. Protein binding reached equilibrium within ~1h of incubation at 37°C. The samples were extracted in turn with: isooctane to collect the unbound fraction; 2-octanol to extract the lipoprotein-bound fraction; and acetonitrile to obtain the protein-bound fraction. Absolute recoveries of DLM, CIS and TRANS ranged from 86 to 95%. Adherence of these very lipophilic chemicals to glass and plastic was minimized by using silanized glass vials and LoBind® plastic pipettes. The method's ability to distinguish lipoprotein from protein binding was confirmed by experiments with diazepam and cyclosporine, drugs that bind selectively to albumin and lipoproteins, respectively. This procedure was effectively utilized for studies of the species-dependence of plasma protein and lipoprotein binding of three pyrethroids for inclusion in physiologically-based pharmacokinetic models of pyrethroids for use in health risk assessments of the insecticides in children and adults. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    NARCIS (Netherlands)

    Koppelman, S. J.; van't Veer, C.; Sixma, J. J.; Bouma, B. N.

    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

  18. CRTC2 Is a Coactivator of GR and Couples GR and CREB in the Regulation of Hepatic Gluconeogenesis.

    Science.gov (United States)

    Hill, Micah J; Suzuki, Shigeru; Segars, James H; Kino, Tomoshige

    2016-01-01

    Glucocorticoid hormones play essential roles in the regulation of gluconeogenesis in the liver, an adaptive response that is required for the maintenance of circulating glucose levels during fasting. Glucocorticoids do this by cooperating with glucagon, which is secreted from pancreatic islets to activate the cAMP-signaling pathway in hepatocytes. The cAMP-response element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) is a coactivator known to be specific to CREB and plays a central role in the glucagon-mediated activation of gluconeogenesis in the early phase of fasting. We show here that CRTC2 also functions as a coactivator for the glucocorticoid receptor (GR). CRTC2 strongly enhances GR-induced transcriptional activity of glucocorticoid-responsive genes. CRTC2 physically interacts with the ligand-binding domain of the GR through a region spanning amino acids 561-693. Further, CRTC2 is required for the glucocorticoid-associated cooperative mRNA expression of the glucose-6-phosphatase, a rate-limiting enzyme for hepatic gluconeogenesis, by facilitating the attraction of GR and itself to its promoter region already occupied by CREB. CRTC2 is required for the maintenance of blood glucose levels during fasting in mice by enhancing the GR transcriptional activity on both the G6p and phosphoenolpyruvate carboxykinase (Pepck) genes. Finally, CRTC2 modulates the transcriptional activity of the progesterone receptor, indicating that it may influence the transcriptional activity of other steroid/nuclear receptors. Taken together, these results reveal that CRTC2 plays an essential role in the regulation of hepatic gluconeogenesis through coordinated regulation of the glucocorticoid/GR- and glucagon/CREB-signaling pathways on the key genes G6P and PEPCK.

  19. New fluorescent reagents specific for Ca2+-binding proteins

    International Nuclear Information System (INIS)

    Ben-Hail, Danya; Lemelson, Daniela; Israelson, Adrian; Shoshan-Barmatz, Varda

    2012-01-01

    Highlights: ► New reagents specifically inhibit the activity of Ca 2+ -dependent proteins. ► FITC-Ru and EITC-Ru allow for mechanism-independent probing of Ca 2+ -binding proteins. ► Changes in reagents fluorescence allow characterization of protein Ca 2+ -binding properties. -- Abstract: Ca 2+ carries information pivotal to cell life and death via its interactions with specific binding sites in a protein. We previously developed a novel photoreactive reagent, azido ruthenium (AzRu), which strongly inhibits Ca 2+ -dependent activities. Here, we synthesized new fluorescent ruthenium-based reagents containing FITC or EITC, FITC-Ru and EITC-Ru. These reagents were purified, characterized and found to specifically interact with and markedly inhibit Ca 2+ -dependent activities but not the activity of Ca 2+ -independent reactions. In contrast to many reagents that serve as probes for Ca 2+ , FITC-Ru and EITC-Ru are the first fluorescent divalent cation analogs to be synthesized and characterized that specifically bind to Ca 2+ -binding proteins and inhibit their activity. Such reagents will assist in characterizing Ca 2+ -binding proteins, thereby facilitating better understanding of the function of Ca 2+ as a key bio-regulator.

  20. Identification of Putative Vero Cell Protein(s) that Bind Specifically to ...

    African Journals Online (AJOL)

    Results: The 45 KDa, 43 KDa and 30 KDa plasma membrane proteins were identified as viral envelope targets. Competitive binding assay showed these proteins competing with dengue virus binding. MTT assay indicate that viability of vero cells increases in cultures pretreated with 45 KDa, 43 KDa and 30 KDa proteins ...

  1. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

    Full Text Available The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs. A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

  2. Water-binding of protein particles

    NARCIS (Netherlands)

    Peters, J.P.C.M.

    2016-01-01

    As overweight and obesity become more prevalent in society, the demand for food products that can help maintain body weight increases. One way to make such products is by decreasing the protein and fat content through increasing the water content. This thesis describes the potential of protein

  3. Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins.

    Science.gov (United States)

    Holth, L T; Sun, J M; Coutts, A S; Murphy, L C; Davie, J R

    1997-12-01

    The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes.

  4. Binding mechanisms of intrinsically disordered proteins: theory, simulation, and experiment

    Directory of Open Access Journals (Sweden)

    Luca Mollica

    2016-09-01

    Full Text Available In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs. In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed e.g. in the fly-casting hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit, are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.

  5. Binding and measuring natural rubber latex proteins on glove powder.

    Science.gov (United States)

    Tomazic-Jezic, Vesna J; Lucas, Anne D; Sanchez, Beatriz A

    2004-01-01

    Cornstarch used as a donning powder on natural rubber latex (NRL) gloves adsorbs NRL proteins. During glove use, powder-carried proteins can be aerosolized and can cause allergic reactions in NRL sensitized individuals. The amount of NRL proteins bound to glove powder and its relative relationship to the total amount of proteins on the glove has not been studied, due to the difficulty in measuring proteins on powder. Using the ELISA inhibition assay for NRL proteins [Standard test method for the immunological measurement of antigenic protein in natural rubber and its products. In: The Annual Book of ASTM Standards; ASTM: West Conshohocken, PA, 2000; ASTM D 64-0] we have investigated possible protocol modifications in order to include measurement of proteins bound to glove powder, as well as the water-extractable glove proteins. Possible interference of the starch itself was evaluated by adding clean cornstarch to the assay. No significant interference was observed with powder concentrations below 5 mg/mL. We analyzed 19 extracts of powdered surgical and examination gloves before and after removal of the particulate component. Comparison of NRL glove extracts with, and without, the cornstarch powder fraction indicated significant variations in the ratios of powder-bound protein and corresponding water-extractable protein. The ratios did not appear to correlate with either the total protein on the glove, the glove weight, or the total amount of powder on the glove. However, when virgin glove powders were exposed to NRL proteins, binding was proportional to the protein concentration in the suspension. Temperature in the range from 4 degrees C to 37 degrees C, did not affect binding intensity, while a higher pH resulted in a higher level of protein associated with, or bound to, the starch. The major differences in the propensity for NRL protein binding were observed among different glove powders. The data indicate that the amount of protein that binds to glove powder

  6. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  7. Phosphodiesterase-4 inhibitors ameliorates cognitive deficits in deoxycorticosterone acetate induced hypertensive rats via cAMP/CREB signaling system.

    Science.gov (United States)

    Jabaris, S Sugin Lal; Sumathy, Haridass; Girish, Ramesh; Narayanan, Shridhar; Sugumar, Mani; Saravana Babu, Chidambaram; Thanikachalam, Sadagopan; Thanikachalam, Mohan

    2015-10-05

    Phosphodiesterase-4 (PDE-4) inhibitors promote memory by blocking the degradation of cAMP. Existing evidence also shows that neuronal survival and plasticity are dependent on the phosphorylation of cAMP-response element-binding protein. In this regard, PDE-4 inhibitors have also been shown to reverse pharmacologically and genetically induced memory impairment in animal models. In the present study, the authors examined the effect of both rolipram and roflumilast (PDE-4 inhibitors) on the impairment of learning and memory observed in hypertensive rats. Deoxycorticosterone acetate (DOCA) salt hypertensive model was used to induce learning and memory deficits. The mRNA expression of different PDE-4 subtypes along with the protein levels of pCREB and BDNF in the hippocampus was quantified. Systolic blood pressure was significantly increased in DOCA salt hypertensive rats when compared to sham operated rats. This effect was reversed by clonidine, an α2 receptor agonist, while PDE-4 inhibitors did not. PDE-4 inhibitors significantly improved the time-induced memory deficits in object recognition task (ORT). In DOCA salt hypertensive rats, the gene expression of PDE-4B and PDE-4D was significantly increased. Furthermore, both pCREB and BDNF showed decreased levels of expression in hypertensive rats in comparison to sham operated rats. Repeated administration of PDE-4 inhibitors significantly decreased both PDE-4B and PDE-4D with an increase in the expression of pCREB and BDNF in hypersensitive rats. Also, rolipram, roflumilast and roflumilast N-oxide showed a linear increase in the plasma and brain concentrations after ORT. Our present findings suggested that PDE-4 inhibitors ameliorate hypertension-induced learning impairment via cAMP/CREB signaling that regulates BDNF expression downstream in the rat hippocampus. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties......Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including...... endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases...

  9. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

    deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

    Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

  10. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

    Flock, Tilman; Hauser, Alexander S; Lund, Nadia

    2017-01-01

    The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of ami...

  11. Long-term neurocognitive dysfunction in offspring via NGF/ ERK/CREB signaling pathway caused by ketamine exposure during the second trimester of pregnancy in rats.

    Science.gov (United States)

    Li, Yanan; Li, Xinran; Guo, Cen; Li, Lina; Wang, Yuxin; Zhang, Yiming; Chen, Yu; Liu, Wenhan; Gao, Li

    2017-05-09

    Early life exposure to ketamine caused neurohistopathologic changes and persistent cognitive dysfunction. For this study, a pregnant rat model was developed to investigate neurocognitive effects in the offspring, following ketamine exposure during the second trimester. Pregnant rats on gestational day 14 (equal to midtrimester pregnancy in humans), intravenously received 200 mg/kg ketamine for 3 h. Their behavior was tested (Morris water maze, odor recognition test, and fear conditioning) at postnatal days (P25-30). Furthermore, hippocampal morphology of the offspring (P30) was examined via Nissl staining and hippocampal dendritic spine density was determined via Golgi staining. The hippocampal protein levels of nerve growth factor (NGF), extracellular signal-regulated kinase (ERK), phosphorylated-ERK (p-ERK), cyclic adenosine monophosphate response element-binding (CREB), p-CREB, synaptophysin (SYP), synapsin (SYN), and postsynaptic density-95 (PSD95) were measured via western blot. Additionally, SCH772984 (an ERK inhibitor) was used to evaluate both role and underlying mechanism of the ERK pathway in PC12 cells. We found that ketamine caused long-term neurocognitive dysfunction, reduced the density of the dendritic spin, caused neuronal loss, and down-regulated the expression of NGF, ERK, p-ERK, mitogen, and stress-activated protein kinase (MSK), CREB, p-CREB, SYP, SYN, and PSD95 in the hippocampus. These results suggest that ketamine induced maternal anesthesia during period of the fetal brain development can cause long-term neurocognitive dysfunction in the offspring, which likely happens via inhibition of the NGF-ERK-CREB pathway in the hippocampus. Our results highlight the central role of ERK in neurocognition.

  12. cAMP/PKA-CREB-BDNF signaling pathway in hippocampus mediates cyclooxygenase 2-induced learning/memory deficits of rats subjected to chronic unpredictable mild stress.

    Science.gov (United States)

    Luo, Ying; Kuang, Shengnan; Li, Huan; Ran, Dongzhi; Yang, Junqing

    2017-05-30

    To investigate the mechanism of cyclooxygenase 2 (COX2) in learning and memory impairments in rats subjected to chronic unpredictable mild stress (CUMS), meloxicam was used intragastrically to inhibit the activity of cyclooxygenase 2. Moreover, cyclooxygenase 2 over-expressing or RNA interfere lentivirus was injected intraventricularly to increase or decrease the enzyme's expression, respectively. The body weights and sucrose consumption were used to analyze depressive behaviors, while the Morris water maze and step-down-type passive avoidance tests were carried out to evaluate the learning-memory functions. The levels of inflammatory cytokines were measured to estimate inflammation and the contents of cyclic adenosine monophosphate (cAMP) were used to measure the levels of the second messenger. Changes in cyclooxygenase 2 mRNA levels were analyzed using reverse transcription polymerase chain reaction. Moreover, the expression of cyclooxygenase 2, brain-derived neurotrophic factor (BDNF), prostaglandins receptor 3 (EP3), protein kinase A (PKA), cAMP response element binding protein (CREB), and phosphorylated CREB were estimated using immunohistochemical staining or western blotting. The results showed that CUMS led to significant depressive-like behaviors and learning and memory dysfunctions. Also, the cAMP levels decreased significantly, while levels of inflammatory cytokines and prostaglandins E2 increased significantly. The expressions of PKA, BDNF, phosphorylated CREB/CREB declined and cyclooxygenase 2 was increased. Meloxicam and cyclooxygenase 2 RNA interfere lentivirus reversed the changes caused by CUMS while cyclooxygenase 2-overexpressing lentivirus worsened these abnormalities. The findings also showed that CUMS increased cyclooxygenase 2 expression, which can cause learning and memory impairments, mainly through activating the hippocampal neuronal cAMP/PKA-CREB-BDNF signaling pathways.

  13. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    modulator of the GABAA receptor in brain membranes. ACBP/DBI, or proteolytically derived polypeptides of ACBP/DBI, have also been implicated in the control of steroidogenesis in mitochondria and glucose-stimulated insulin secretion. Thus, it appears that ACBP/DBI is a remarkable, versatile protein. Now we......Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous....... There is a remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  14. Detergent activation of the binding protein in the folate radioassay

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  15. RNA-Binding Domain Proteins in Kinetoplastids: a Comparative Analysis†

    Science.gov (United States)

    De Gaudenzi, Javier; Frasch, Alberto C.; Clayton, Christine

    2005-01-01

    RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein. PMID:16339728

  16. Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

    Science.gov (United States)

    Haupt, V Joachim; Daminelli, Simone; Schroeder, Michael

    2013-01-01

    Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor

  17. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein

    NARCIS (Netherlands)

    Hateboer, G.; Timmers, H.T.M.; Rustgi, A.K.; Billaud, Marc; Veer, L.J. Van 't; Bernards, R.A.

    1993-01-01

    Using a protein binding assay, we show that the amino-teminal 204 amino acids of the c-Myc protein interact di y with a key component of the basal p tdon factor TFID, the TATA box-binding protein (TBP). Essentialy the same region of the c-Myc protein alo binds the product of the retinoblatoma

  18. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

    Science.gov (United States)

    Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

    2008-04-01

    Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

  19. Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

    Science.gov (United States)

    Bach, Julia; Lewis, Nathaniel; Maggiora, Kathy; Gillespie, Alison J; Connell-Crowley, Lisa

    2015-08-28

    This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  1. Computational design of binding proteins to EGFR domain II.

    Directory of Open Access Journals (Sweden)

    Yoon Sup Choi

    Full Text Available We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.

  2. CAG trinucleotide RNA repeats interact with RNA-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, B.A.; Eberwine, J.; Spencer, C. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    1996-09-01

    Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington`s disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to >37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and LJV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases. 47 refs., 5 figs.

  3. CNE, a collagen-binding protein of Streptococcus equi.

    Science.gov (United States)

    Lannergård, Jonas; Frykberg, Lars; Guss, Bengt

    2003-05-16

    Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virulence factor in septic arthritis. Based on similarity to CNA the structure of the mature CNE protein can be divided into an N-terminal A domain and a C-terminal B domain. The highest similarity between CNA and CNE is found in the A domains. The A domain in CNA is known to be the collagen-binding domain. Two parts of cne were amplified using polymerase chain reaction (PCR) and ligated into an expression vector, and recombinant CNE proteins were produced in Escherichia coli. The purified CNE proteins were shown to display collagen-binding activity in a Western ligand blot and to inhibit collagen binding to cells of subsp. equi and to CNE-coated microtitre wells. Furthermore, the A domain of CNE was sufficient for binding collagen, and was shown to compete for the same site on collagen as CNA in inhibition studies. Using PCR, the cne gene was detected in all studied strains of subsp. equi and S. equi subsp. zooepidemicus.

  4. Developmental iodine deficiency resulting in hypothyroidism reduces hippocampal ERK1/2 and CREB in lactational and adolescent rats

    Science.gov (United States)

    2009-01-01

    Background Developmental iodine deficiency (ID) leads to inadequate thyroid hormone that impairs learning and memory with an unclear mechanism. Here, we show that hippocampal extracellular signal-regulated kinase (ERK1/2) and cAMP response element-binding protein (CREB) are implicated in the impaired learning and memory in lactational and adolescent rat hippocampus following developmental ID and hypothyroidism. Methods Three developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil (PTU, 5 ppm or 15 ppm)-added drinking water from gestational day (GD) 6 till postnatal day (PN) 28. Then, the total and phorsporylated ERK1/2 and total and phorsporylated CREB in the hippocampus were detected with western blot on PN14, PN21, PN28 and PN42. Results The iodine-deficient and hypothyroid pups showed lower serum FT3 and FT4 levels, smaller body size, and delayed eyes opening. The mean number of surviving cells in the hippocampus of the iodine-deficient and 15 ppm PTU-treated rats was significantly reduced compared to controls (P hypothyroidism down-regulate hippocampal ERK1/2 and CREB in lactational and adolescent rats. PMID:20021662

  5. Sleep deprivation prevents stimulation-induced increases of levels of P-CREB and BDNF: protection by caffeine.

    Science.gov (United States)

    Alhaider, Ibrahim A; Aleisa, Abdulaziz M; Tran, Trinh T; Alkadhi, Karim A

    2011-04-01

    It is well known that caffeine and sleep deprivation have opposing effects on learning and memory; therefore, this study was undertaken to determine the effects of chronic (4wks) caffeine treatment (0.3g/l in drinking water) on long-term memory deficit associated with 24h sleep deprivation. Animals were sleep deprived using the modified multiple platform method. The results showed that chronic caffeine treatment prevented the impairment of long-term memory as measured by performance in the radial arm water maze task and normalized L-LTP in area CA1 of the hippocampi of sleep-deprived anesthetized rats. Sleep deprivation prevents the high frequency stimulation-induced increases in the levels of phosphorylated-cAMP response element binding protein (P-CREB) and brain-derived neurotrophic factor (BDNF) seen during the expression of late phase long-term potentiation (L-LTP). However, chronic caffeine treatment prevented the effect of sleep-deprivation on the stimulated levels of P-CREB and BDNF. The results suggest that chronic caffeine treatment may protect the sleep-deprived brain probably by preserving the levels of P-CREB and BDNF. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G.

    Science.gov (United States)

    Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

    2017-01-01

    The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm -positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis , respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis . The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.

  7. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  8. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. © 2014 The Authors.

  9. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  10. Epigenetic modification of miR-10a regulates renal damage by targeting CREB1 in type 2 diabetes mellitus.

    Science.gov (United States)

    Shan, Qun; Zheng, Guihong; Zhu, Aihua; Cao, Li; Lu, Jun; Wu, Dongmei; Zhang, ZiFeng; Fan, Shaohua; Sun, Chunhui; Hu, Bin; Zheng, Yuanlin

    2016-09-01

    Emerging evidence has shown that microRNA-mediated gene expression modulation plays a crucial role in the pathogenesis of type 2 diabetes mellitus, but the novel miRNAs involved in type 2 diabetes and its functional regulatory mechanisms still need to be determined. In this study, we assessed the role of miR-10a in extracellular matrix accumulation in the kidney of diabetic mellitus induced by combining administration of chronic high fat diet (HFD) and low dosage of streptozotocin (STZ, 35mg/kg). Here, we found that HFD/STZ administration decreased the level of microRNA (miR-10a) expression in ICR strain mice. Overexpression of miR-10a alleviated the increased ratio of urine albumin-to-creatinine (ACR) ratio of HFD/STZ mice. In contrast, knockdown of miR-10a increased the ratio of kidney ACR in naïve mice. Furthermore, cAMP response element binding protein 1 (CREB1) was validated as a target of miR-10a in vitro and in vivo. CREB1 and its downstream fibronectin (FN, extracellular matrix) were increased in HFD/STZ-treated mice, which was reversed by kidney miR-10a overexpression. The content of CREB1 and FN was increased by miR-10a knockdown in kidney of naïve mice. Furthermore, histone deacetylase 3 (HDAC3) was revealed to be increased in kidney of HFD/STZ mice, accompanied with the augmentation of ACR ratio and FN level. Knockdown of HDAC3 with siRNA significantly caused the increase of miR-10a, resulting in the decrease in CREB1 and FN expression in kidney of HFD/STZ mice. Contrarily, HDAC3 overexpression mediated by lentivirus decreased miR-10a content, and enhanced ACR value, CREB1 and FN formation in naïve mice. Collectively, these results elucidate that HDAC3/miR-10a/CREB1 serves as a new mechanism underlying kidney injury, providing potential therapeutic targets in type 2 diabetes. Copyright © 2016. Published by Elsevier Inc.

  11. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  12. T3: Targeted Proteomics of DNA-Binding Proteins

    OpenAIRE

    Nagore, Linda I.; Jarrett, Harry W.

    2015-01-01

    A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TF) while providing a route for high throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TF by combining nuclear extract with a specific DNA sequence and imm...

  13. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  14. Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells.

    Science.gov (United States)

    Huang, Hao; Hong, Qian; Tan, Hong-Ling; Xiao, Cheng-Rong; Gao, Yue

    2016-12-01

    Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells. PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA. Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10-40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2. The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS-induced up-regulation of PDE4B and

  15. Chromate Binding and Removal by the Molybdate-Binding Protein ModA.

    Science.gov (United States)

    Karpus, Jason; Bosscher, Michael; Ajiboye, Ifedayo; Zhang, Liang; He, Chuan

    2017-04-04

    Effective and cheap methods and techniques for the safe removal of hexavalent chromate from the environment are in increasingly high demand. High concentrations of hexavalent chromate have been shown to have numerous harmful effects on human biology. We show that the E. coli molybdate-binding protein ModA is a genetically encoded tool capable of removing chromate from aqueous solutions. Although previously reported to not bind chromate, we show that ModA binds chromate tightly and is capable of removing chromate to levels well below current US federal standards. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

    Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F; Schiller, Tara; Musumeci, Anthony; Martin, Darren

    2009-01-01

    Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO 2 , the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO 2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

  18. Solvation structure of ice-binding antifreeze proteins

    Science.gov (United States)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  19. CREB pathway links PGE2 signaling with macrophage polarization.

    Science.gov (United States)

    Luan, Bing; Yoon, Young-Sil; Le Lay, John; Kaestner, Klaus H; Hedrick, Susan; Montminy, Marc

    2015-12-22

    Obesity is thought to promote insulin resistance in part via activation of the innate immune system. Increases in proinflammatory cytokine production by M1 macrophages inhibit insulin signaling in white adipose tissue. In contrast, M2 macrophages have been found to enhance insulin sensitivity in part by reducing adipose tissue inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP pathway, although the underlying mechanism is unclear. Here we show that PGE2 stimulates M2 polarization via the cyclic AMP-responsive element binding (CREB)-mediated induction of Krupple-like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages down-regulated M2 marker gene expression and promoted insulin resistance in the context of high-fat diet feeding. As re-expression of KLF4 rescued M2 marker gene expression in CREB-depleted cells, our results demonstrate the importance of the CREB/CRTC pathway in maintaining insulin sensitivity in white adipose tissue via its effects on the innate immune system.

  20. Phosphorus Binding Sites in Proteins: Structural Preorganization and Coordination

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Greisen, Per Junior; Junker, Märta Caroline

    2014-01-01

    to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced...... by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell...

  1. Ectopic Phosphorylated Creb Marks Dedifferentiated Proximal Tubules in Cystic Kidney Disease.

    Science.gov (United States)

    Puri, Pawan; Schaefer, Caitlin M; Bushnell, Daniel; Taglienti, Mary E; Kreidberg, Jordan A; Yoder, Bradley K; Bates, Carlton M

    2018-01-01

    Ectopic cAMP signaling is pathologic in polycystic kidney disease; however, its spatiotemporal actions are unclear. We characterized the expression of phosphorylated Creb (p-Creb), a target and mediator of cAMP signaling, in developing and cystic kidney models. We also examined tubule-specific effects of cAMP analogs in cystogenesis in embryonic kidney explants. In wild-type mice, p-Creb marked nephron progenitors (NP), early epithelial NP derivatives, ureteric bud, and cortical stroma; p-Creb was present in differentiated thick ascending limb of Henle, collecting duct, and stroma; however, it disappeared in mature NP-derived proximal tubules. In Six2cre;Frs2α Fl/Fl mice, a renal cystic model, ectopic p-Creb stained proximal tubule-derived cystic segments that lost the differentiation marker lotus tetragonolobus lectin. Furthermore, lotus tetragonolobus lectin-negative/p-Creb-positive cyst segments (re)-expressed Ncam1, Pax2, and Sox9 markers of immature nephron structures and dedifferentiated proximal tubules after acute kidney injury. These dedifferentiation markers were co-expressed with p-Creb in renal cysts in Itf88 knockout mice subjected to ischemia and Six2cre;Pkd1 Fl/Fl mice, other renal cystogenesis models. 8-Br-cAMP addition to wild-type embryonic kidney explants induced proximal tubular cystogenesis and p-Creb expression; these effects were blocked by co-addition of protein kinase A inhibitor. Thus p-Creb/cAMP signaling is appropriate in NP and early nephron derivatives, but disappears in mature proximal tubules. Moreover, ectopic p-Creb expression/cAMP signaling marks dedifferentiated proximal tubular cystic segments. Furthermore, proximal tubules are predisposed to become cystic after cAMP stimulation. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena [Institute for Health and Consumer Protection (IHCP), JRC, Ispra (Italy); Accordi, Benedetta; Basso, Giuseppe [Oncohematology Laboratory, Department of Woman and Child Health, University of Padova, Padova (Italy); Gaspar, John Antonydas [Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, Cologne (Germany); Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina [Institute for Health and Consumer Protection (IHCP), JRC, Ispra (Italy); Sachinidis, Agapios [Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, Cologne (Germany); Bremer-Hoffmann, Susanne, E-mail: susanne.bremer@jrc.ec.europa.eu [Institute for Health and Consumer Protection (IHCP), JRC, Ispra (Italy)

    2014-10-15

    According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.

  3. Effects of ketamine administration on the phosphorylation levels of CREB and TrKB and on oxidative damage after infusion of MEK inhibitor.

    Science.gov (United States)

    Réus, Gislaine Z; Abaleira, Helena M; Titus, Stephanie E; Arent, Camila O; Michels, Monique; da Luz, Jaine R; dos Santos, Maria Augusta B; Carlessi, Anelise S; Matias, Beatriz I; Bruchchen, Livia; Steckert, Amanda V; Ceretta, Luciane B; Dal-Pizzol, Felipe; Quevedo, João

    2016-02-01

    Ketamine, an antagonist of N-methyl-d-aspartate (NMDA) receptors, has presented antidepressant effects in basic and clinical studies. The MAPK kinase (MEK) signaling pathway could be a target for novel antidepressant drugs and an important pathway involved in neuronal plasticity. Thus, this study evaluated the effects of the administration of ketamine on the phosphorylation of TrKB and CREB, and oxidative stress parameters in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens (NAc) rats, after the inhibition of MAPK pathway (PD184161). Male adult Wistar rats were submitted to a surgical procedure to receive a single dose of a pharmacological inhibitor of MAPK (PD184161) at a dose of (0.1μg/μl) or vehicle. Then, they were divided: 1) vehicle+saline; 2) inhibitor PD184161+saline; 3) vehicle+ketamine 15mg/kg; and 4) inhibitor PD184161+ketamine 15mg/kg. MEK inhibitor and ketamine increased the phosphorylation of the transcription factor cAMP response element-binding protein (pCREB) and neurotrophic factor/tropomyosin related kinase B receptor (pTrKB) in the PFC, and decreased pCREB in the hippocampus. The MEK inhibitor abolished ketamine's effects in the hippocampus. In the amygdala, pCREB was decreased, and pTrKB was increased after MEK inhibitor plus ketamine. Ketamine increased the thiobarbituric acid reactive species (TBARS) in the PFC, hippocampus, amygdala, and NAc; MEK inhibitor antagonized these effects. The carbonyl was increased in the PFC by both ketamine and MEK inhibitor, but inhibitor infusion plus ketamine administration reduced this effect. In the amygdala, MEK inhibitor increased carbonyl. Ketamine's effects on pCREB, pTrKB, and oxidative stress are mediated, at least in part, by a mechanism dependent of MAPK signaling inhibition. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  4. Time-dependent co-relation of BDNF and CREB mRNAs in adult rat brains following acute psychological stress in the communication box paradigm.

    Science.gov (United States)

    Li, Gongying; Wang, Yanmei; Yan, Min; Ma, Hongxia; Gao, Yanjie; Li, Zexuan; Li, Changqi; Tian, Hongjun; Zhuo, Chuanjun

    2016-06-15

    Psychological stress affects human health, and chronic stress leads to life-threatening diseases, such as depression and post-traumatic stress disorder. Psychological stress coping mechanisms involve the brain-derived neurotrophic factor (BDNF) and downstream cAMP response element binding protein (CREB), which are targets of the adverse effects of stress paradigms. Fourty-seven adult male Sprague-Dawley rats were divided into control, physical stress and six psychological stress groups which were assayed at 0h, 0.5h, 1h, 2h, 6h and 24h after communication box (CB) stress induction. Behavioral assessment using open field and elevated plus maze tests determined that CB stress significantly increased anxiety. After CB stress, the alternation of mRNA levels of BDNF and CREB were assessed at different time points by in situ hybridization. The mRNA levels of BDNF and CREB were significantly decreased, then gradually recovered over 24h to maximum levels in the hippocampus (CA1 region), prefrontal cortex (PFC), central amygdaloid nuclei (AG), shell of accumbens nucleus (NAC), periaqueductal gray (PAG) and ventral tegmental area, except for the ventral tegmental area (VTA). Moreover, mRNA levels of BDNF and CREB were positively correlated in all examined brain regions, except for the VTA region at 0 and 24h after CB stress induction. These findings suggest that BDNF and CREB may belong to the same pathway and be involved in psychological stress response mechanisms, and protect the organism from stress induced, aversive processes leading to disease. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  5. The Traditional Japanese Herbal Medicine Hachimijiogan Elicits Neurite Outgrowth Effects in PC12 Cells and Improves Cognitive in AD Model Rats via Phosphorylation of CREB

    Directory of Open Access Journals (Sweden)

    Kaori Kubota

    2017-11-01

    Full Text Available Hachimijiogan (HJG is a traditional herbal medicine that improves anxiety disorders in patients with dementia. In this study, we tested the hypothesis that HJG exerts neurotrophic factor-like effects to ameliorate memory impairment in Alzheimer disease (AD model rats. First, we describe that HJG acts to induce neurite outgrowth in PC12 cells (a rat pheochromocytoma cell line like nerve growth factor (NGF in a concentration-dependent manner (3 μg/ml HJG, p < 0.05; 10–500 μg/ml HJG, p < 0.001. While six herbal constituents of HJG, Rehmannia root, Dioscorea rhizome, Rhizoma Alismatis, Poria sclerotium, Moutan bark, and Cinnamon bark, could induce neurite outgrowth effects, the effect was strongest with HJG (500 μg/ml. Second, we demonstrated that HJG-induced neurite outgrowth was blocked by an inhibitor of cAMP response element binding protein (CREB, KG-501 (10 μM, p < 0.001. Moreover, HJG was observed to induce CREB phosphorylation 20–90 min after treatment (20 min, 2.50 ± 0.58-fold and CRE-mediated transcription in cultured PC12 cells (500 μg/ml, p < 0.01; 1000 μg/ml, p < 0.001. These results suggest a CREB-dependent mechanism underlies the neurotrophic effects of HJG. Finally, we examined improvements of memory impairment following HJG treatment using a Morris water maze in AD model animals (CI + Aβ rats. Repeated oral administration of HJG improved memory impairment (300 mg/kg, p < 0.05; 1000 mg/kg, p < 0.001 and induced CREB phosphorylation within the hippocampus (1000 mg/kg, p < 0.01. Together, our results suggest that HJG possesses neurotrophic effects similar to those of NGF, and can ameliorate cognitive dysfunction in a rat dementia model via CREB activation. Thus, HJG could potentially be a substitute for neurotrophic factors as a treatment for dementia.

  6. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  7. CREB coactivators CRTC2 and CRTC3 modulate bone marrow hematopoiesis.

    Science.gov (United States)

    Kim, Jeong-Ho; Hedrick, Susan; Tsai, Wen-Wei; Wiater, Ezra; Le Lay, John; Kaestner, Klaus H; Leblanc, Mathias; Loar, Andrew; Montminy, Marc

    2017-10-31

    Populations of circulating immune cells are maintained in equilibrium through signals that enhance the retention or egress of hematopoietic stem cells (HSCs) from bone marrow (BM). Prostaglandin E2 (PGE2) stimulates HSC renewal and engraftment through, for example, induction of the cAMP pathway. Triggering of PGE2 receptors increases HSC survival in part via the PKA-mediated induction of the cAMP response element-binding protein (CREB) signaling pathway. PKA stimulates cellular gene expression by phosphorylating CREB at Ser133 and by promoting the dephosphorylation of the cAMP- responsive transcriptional coactivators (CRTCs). We show here that disruption of both CRTC2 and CRTC3 causes embryonic lethality, and that a single allele of either CRTC2 or CRTC3 is sufficient for viability. CRTC2 knockout mice that express one CRTC3 allele (CRTC2/3m mice) develop neutrophilia and splenomegaly in adulthood due to the up-regulation of granulocyte-colony stimulating factor (G-CSF); these effects are reversed following administration of neutralizing anti-G-CSF antiserum. Adoptive transfer of CRTC2/3m BM conferred the splenomegaly/neutrophilia phenotype in WT recipients. Targeted disruption of both CRTC2 and CRTC3 in stromal cells with a mesenchymal Prx1-Cre transgene also promoted this phenotype. Depletion of CRTC2/3 was found to decrease the expression of Suppressor of Cytokine Signaling 3 (SOCS3), leading to increases in STAT3 phosphorylation and to the induction of CEBPβ, a key regulator of the G-CSF gene. As small molecule inhibition of JAK activity disrupted CEBPβ induction and reduced G-CSF expression in CRTC2/3m stromal cells, our results demonstrate how cross-coupling between the CREB/CRTC and JAK/STAT pathways contributes to BM homeostasis. Published under the PNAS license.

  8. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... the same patients exhibited little immunoreactivity. Both the peptide antiserum and the polyclonal antiserum against the native protein immunoblotted a molecular weight 63,000 protein in nuclear extracts of tumor tissue, but not significantly in extracts of normal tissue. At the molecular level......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

  9. Free enthalpies of replacing water molecules in protein binding pockets.

    Science.gov (United States)

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  10. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E. (Lawrence Berkeley Lab., CA (United States))

    1991-06-04

    Tritium-labeled {alpha}- and {beta}-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10{degrees}C, MBP bound {alpha}-maltose with 2.7 {plus minus} 0.5-fold higher affinity than {beta}-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound {alpha}-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound {beta}-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the {beta}-maltodextrin is bound by its reducing end, and, in the other complex, the {beta}-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

  11. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    International Nuclear Information System (INIS)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

    1991-01-01

    Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

  12. Fluorescence properties of porcine odorant binding protein Trp 16 residue

    Energy Technology Data Exchange (ETDEWEB)

    Albani, Jihad Rene, E-mail: Jihad-Rene.Albani@univ-lille1.f [Laboratoire de Biophysique Moleculaire, Universite des Sciences et Technologies de Lille, F-59655 Villeneuve d' Ascq Cedex (France)

    2010-11-15

    Summary: The present work deals with fluorescence studies of adult porcine odorant binding protein at pH=7.5. At this pH, the protein is a dimer, each monomer contains one tryptophan residue. Our results show that tryptophan residue displays significant motions and emits with three fluorescence lifetimes. Decay associated spectra showed that the three lifetime's components emanate from sub-structures surrounded by the same microenvironment.

  13. SCM-198 Ameliorates Cognitive Deficits, Promotes Neuronal Survival and Enhances CREB/BDNF/TrkB Signaling without Affecting Aβ Burden in AβPP/PS1 Mice.

    Science.gov (United States)

    Hong, Zhen-Yi; Yu, Shuang-Shuang; Wang, Zhi-Jun; Zhu, Yi-Zhun

    2015-08-07

    SCM-198 is an alkaloid found only in Herba leonuri and it has been reported to possess considerable neuroprotective effects in animal models of ischemic stroke, Parkinson's disease and Alzheimer's disease (AD). In this study, we demonstrated for the first time that 3-month oral SCM-198 treatment could significantly improve both recognition and spatial memory, inhibit microgliosis and promote neuronal survival in amyloid-β protein precursor and presenilin-1(AβPP/PS1) double-transgenic mice without affecting amyloid-β (Aβ) burden. In addition, decreases in cAMP-response element-binding protein (CREB) phosphorylation, brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) phosphorylation were attenuated by SCM-198 both in vivo and in primary cortical neurons, which could be blocked by protein kinase A (PKA) inhibitors, suggesting the involvement of upstream PKA in enhancing the BDNF/TrkB/CREB signaling by SCM-198. Our results indicate that SCM-198, a drug that could promote neuronal survival and enhance BDNF/TrkB/CREB signaling, has beneficial effects on behavioral and biochemical alterations without affecting Aβ burden in AβPP/PS1 mice and might become a potential drug candidate for AD treatment in the future.

  14. SCM-198 Ameliorates Cognitive Deficits, Promotes Neuronal Survival and Enhances CREB/BDNF/TrkB Signaling without Affecting Aβ Burden in AβPP/PS1 Mice

    Directory of Open Access Journals (Sweden)

    Zhen-Yi Hong

    2015-08-01

    Full Text Available SCM-198 is an alkaloid found only in Herba leonuri and it has been reported to possess considerable neuroprotective effects in animal models of ischemic stroke, Parkinson’s disease and Alzheimer’s disease (AD. In this study, we demonstrated for the first time that 3-month oral SCM-198 treatment could significantly improve both recognition and spatial memory, inhibit microgliosis and promote neuronal survival in amyloid-β protein precursor and presenilin-1(AβPP/PS1 double-transgenic mice without affecting amyloid-β (Aβ burden. In addition, decreases in cAMP-response element-binding protein (CREB phosphorylation, brain-derived neurotrophic factor (BDNF and tropomyosin-related kinase B (TrkB phosphorylation were attenuated by SCM-198 both in vivo and in primary cortical neurons, which could be blocked by protein kinase A (PKA inhibitors, suggesting the involvement of upstream PKA in enhancing the BDNF/TrkB/CREB signaling by SCM-198. Our results indicate that SCM-198, a drug that could promote neuronal survival and enhance BDNF/TrkB/CREB signaling, has beneficial effects on behavioral and biochemical alterations without affecting Aβ burden in AβPP/PS1 mice and might become a potential drug candidate for AD treatment in the future.

  15. Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels.

    Science.gov (United States)

    Williams, Claire M; El Mohsen, Manal Abd; Vauzour, David; Rendeiro, Catarina; Butler, Laurie T; Ellis, Judi A; Whiteman, Matthew; Spencer, Jeremy P E

    2008-08-01

    Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway.

  16. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B

    1999-01-01

    spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...... is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration...

  17. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

  19. Cloning and expression analysis of a blue copper- binding protein ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... Full Length Research Paper. Cloning and expression analysis of a blue copper- binding protein gene from Dasypyrum Villosum. Huagang He1*, Shanying Zhu1, Wenbing Wang1, Tongde Bie2 and Peidu Chen3. 1Jiangsu University. Zhenjiang 212013, P. R. China. 2Yangzhou Academy of Agricultural ...

  20. Lipopolysaccharide (LPS)-binding protein mediates LPS detoxification by chylomicrons

    NARCIS (Netherlands)

    Vreugdenhil, Anita C. E.; Rousseau, Corine H.; Hartung, Thomas; Greve, Jan Willem M.; van 't Veer, Cornelis; Buurman, Wim A.

    2003-01-01

    Chylomicrons have been shown to protect against endotoxin-induced lethality. LPS-binding protein (LBP) is involved in the inactivation of bacterial toxin by lipoproteins. The current study examined the interaction among LBP, chylomicrons, and bacterial toxin. LBP was demonstrated to associate with

  1. MTBindingSim: simulate protein binding to microtubules.

    Science.gov (United States)

    Philip, Julia T; Pence, Charles H; Goodson, Holly V

    2012-02-01

    Many protein-protein interactions are more complex than can be accounted for by 1:1 binding models. However, biochemists have few tools available to help them recognize and predict the behaviors of these more complicated systems, making it difficult to design experiments that distinguish between possible binding models. MTBindingSim provides researchers with an environment in which they can rapidly compare different models of binding for a given scenario. It is written specifically with microtubule polymers in mind, but many of its models apply equally well to any polymer or any protein-protein interaction. MTBindingSim can thus both help in training intuition about binding models and with experimental design. MTBindingSim is implemented in MATLAB and runs either within MATLAB (on Windows, Mac or Linux) or as a binary without MATLAB (on Windows or Mac). The source code (licensed under the GNU General Public License) and binaries are freely available at http://mtbindingsim.googlecode.com. jphilip@nd.edu; cpence@nd.edu.

  2. Genome-wide regulation of TATA-binding protein activity

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

    Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different

  3. Controlling transcription fidelity via TATA-binding protein dynamics

    NARCIS (Netherlands)

    Koster, M.J.E.

    2015-01-01

    Transcription underlies all cellular processes and responses to internal and external cues. Deregulation of transcription has implications for the fitness of the cell or organism. During my PhD I have investigated the importance of proper TATA-binding protein (TBP) regulation as a mechanism to

  4. Evidence for covalent binding of epicocconone with proteins from ...

    Indian Academy of Sciences (India)

    TECS

    Evidence for covalent binding of epicocconone with proteins from synchronous fluorescence spectra ... the interaction of epicocconone with human serum albumin is significantly different from its interaction with surfactant assemblies. .... at 620 nm is collected at right angles to the direction of the excitation beam, at magic ...

  5. Immunoglobulin classes, metal binding proteins, and trace metals in ...

    African Journals Online (AJOL)

    , IgA and IgM), metal binding proteins (Transferrin, Caeruloplasmin, Alpha-2- Macroglobulin and Haptoglobin) and nutritionally essential trace metals/heavy metals (Zn, Fe, Se, Cu, Mg, Cd and Pb) in Nigerian cassava processors using single ...

  6. molecular interactions of the TATA-binding protein

    Indian Academy of Sciences (India)

    Unknown

    variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the.

  7. PRODIGY: a web server for predicting the binding affinity of protein-protein complexes.

    Science.gov (United States)

    Xue, Li C; Rodrigues, João Pglm; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-12-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given protein-protein complex. Here we present PROtein binDIng enerGY prediction (PRODIGY), a web server to predict the binding affinity of protein-protein complexes from their 3D structure. The PRODIGY server implements our simple but highly effective predictive model based on intermolecular contacts and properties derived from non-interface surface. PRODIGY is freely available at: http://milou.science.uu.nl/services/PRODIGY CONTACT: a.m.j.j.bonvin@uu.nl, a.vangone@uu.nl. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Sweet Dream Liquid Chinese Medicine Ameliorates Learning and Memory Deficit in a Rat Model of Paradoxical Sleep Deprivation through the ERK/CREB Signaling Pathway.

    Science.gov (United States)

    Su, Xinyun; Wang, Chunhua; Wang, Xiuhua; Han, Fang; Lv, Changjun; Zhang, Xiuli

    2016-05-01

    Sweet dream oral liquid (SDOL), a traditional Chinese herbal compound contains 17 traditional Chinese medicines. It has various pharmacological effects, such as improving brain dysfunction and increasing sleeping quality. This study investigated the neuroprotective effect and the underlying mechanisms of SDOL-impaired hippocampus learning and memory-induced paradoxical sleep deprivation (PSD) in rats. Sixty Male Wistar rats were randomly divided into six groups. Before PSD, SDOL treatment group rats were intragastrically administered SDOL for 25 days at dose of 2.1, 4.2, and 8.4 mL/kg body weight per day. Normal control group, large platform control group, and PSD groups were treated with normal saline instead of SDOL. After 25 days treatment, PSD and SDOL groups were deprived of paradoxical sleep for 72 h. Then two behavioral studies were conducted to test the spatial learning and memory ability using the open field test and Morris water maze test. Expression of the c-fos, c-jun, cyclic AMP response element binding protein (CREB), extracellular signal-regulated protein kinase (ERK), mitogen-activated protein kinases (MAPK)/ERK kinase (MEK), and p-CREB, p-ERK, and p-MEK in the hippocampus were also assayed by western blot. In this study, PSD decreased the levels of p-CREB, p-ERK, p-MEK, c-fos, and c-jun. However, SDOL treatment increased expressions of these proteins. Our results showed that SDOL improved 72-h PSD-induced cognitive impairment. These affects may be mediated by increasing the contents of c-fos, c-jun, and p-CREB/ERK signaling.

  9. Small world network strategies for studying protein structures and binding.

    Science.gov (United States)

    Taylor, Neil R

    2013-01-01

    Small world network concepts provide many new opportunities to investigate the complex three dimensional structures of protein molecules. This mini-review explores the published literature on using small-world network approaches to study protein structure, with emphasis on the different combinations of descriptors that have been tested, on studies involving ligand binding in protein-ligand complexes, and on protein-protein complexes. The benefits and success of small world network approaches, which change the focus from specific interactions to the local environment, even to non-local phenomenon, are described. The purpose is to show the different ways that small world network concepts have been used for building new computational models for studying protein structure and function, and for extending and improving existing modelling approaches.

  10. Sampling and energy evaluation challenges in ligand binding protein design.

    Science.gov (United States)

    Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David

    2017-12-01

    The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  11. Engineering periplasmic ligand binding proteins as glucose nanosensors

    Directory of Open Access Journals (Sweden)

    Constance J. Jeffery

    2011-01-01

    Full Text Available Diabetes affects over 100 million people worldwide. Better methods for monitoring blood glucose levels are needed for improving disease management. Several labs have previously made glucose nanosensors by modifying members of the periplasmic ligand binding protein superfamily. This minireview summarizes recent developments in constructing new versions of these proteins that are responsive within the physiological range of blood glucose levels, employ new reporter groups, and/or are more robust. These experiments are important steps in the development of novel proteins that have the characteristics needed for an implantable glucose nanosensor for diabetes management: specificity for glucose, rapid response, sensitivity within the physiological range of glucose concentrations, reproducibility, and robustness.

  12. Observation of Protein Structural Vibrational Mode Sensitivity to Ligand Binding

    Science.gov (United States)

    Niessen, Katherine; Xu, Mengyang; Snell, Edward; Markelz, Andrea

    2014-03-01

    We report the first measurements of the dependence of large-scale protein intramolecular vibrational modes on ligand binding. These collective vibrational modes in the terahertz (THz) frequency range (5-100 cm-1) are of great interest due to their predicted relation to protein function. Our technique, Crystals Anisotropy Terahertz Microscopy (CATM), allows for room temperature, table-top measurements of the optically active intramolecular modes. CATM measurements have revealed surprisingly narrowband features. CATM measurements are performed on single crystals of chicken egg-white lysozyme (CEWL) as well as CEWL bound to tri-N-acetylglucosamine (CEWL-3NAG) inhibitor. We find narrow band resonances that dramatically shift with binding. Quasiharmonic calculations are performed on CEWL and CEWL-3NAG proteins with CHARMM using normal mode analysis. The expected CATM response of the crystals is then calculated by summing over all protein orientations within the unit cell. We will compare the CATM measurements with the calculated results and discuss the changes which arise with protein-ligand binding. This work is supported by NSF grant MRI 2 grant DBI2959989.

  13. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  14. Thermal unfolding of a Ca- and Lanthanide-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Goettfert, M. [Technische Univ. Dresden (Germany); Knoeppel, J.

    2017-06-01

    The MIIA (metal ion-induced autocleavage)-domain of the protein Vic001052 from the pathogen Vibrio coralliilyticus, comprises 173 amino acids and exhibits Ca-dependent autoproteolytic activity. It shows homology to nodulation proteins which are secreted by Rhizobiacea into plant host cells where they exert Ca-dependent functions. We have studied the structural and energetic aspects of metal protein interactions of the MIIA domain which appear attractive for engineering metal-binding synthetic peptides. Using a non-cleavable MIIA domain construct, we detected very similar structural changes upon binding to Ca{sup 2+} and Eu{sup 3+}. The thermal denaturation of the Ca-bound state was studied by circular dichroism spectroscopy. The metal-bound folded state unfolds reversibly into an unstructured metal-free state similar to the metal-free state at room temperature.

  15. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that

  16. An angiogenin-binding protein from endothelial cells

    International Nuclear Information System (INIS)

    Hu, Guofu; Chang, Sooik; Riordan, J.F.; Vallee, B.L.

    1991-01-01

    A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. 125 I-Ang can be crosslinked efficiently to AngBP by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. Bovine ribonuclease A competes with the binding of 125 I-Ang to AngBP, but lysozyme does not. Direct binding to AngBP of 125 I-labeled bovine ribonuclease A is, however, much weaker than that of 125 I-Ang. Two enzymatically active derivatives of angiogenin cleaved at residues 60-61 and 67-68, respectively, fail to induce angiogenesis and also bind to AngBP only weakly. AngBP has been isolated by treatment of cells with heparan sulfate, affinity chromatography on angiogenin-Sepharose of the material dissociated from the cell surface, and gel filtration HPLC. The results suggest that AngBP has the characteristics of a receptor that may likely function in angiogenesis

  17. Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.

    Directory of Open Access Journals (Sweden)

    Emi Ishida

    Full Text Available Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30 and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB, without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK kinase inhibitor and an L-type Calcium (Ca(2+ channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons.

  18. Insulin-like growth factor binding proteins: a structural perspective

    Directory of Open Access Journals (Sweden)

    Briony eForbes

    2012-03-01

    Full Text Available Insulin-like growth factor binding proteins (IGFBP-1 to -6 bind insulin-like growth factors-I and -II (IGF-I and IGF-II with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation and survival via the type 1 IGF receptor (IGF-1R. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that influence processes such as cell migration and apoptosis by influencing gene transcription.IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, Linker and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP to date, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarises structural studies reported so far and highlights features important for binding not only IGF but also other partners. It also highlights future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease.

  19. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  20. Sampling protein motion and solvent effect during ligand binding

    Science.gov (United States)

    Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

    2012-01-01

    An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

  1. Binding free energy analysis of protein-protein docking model structures by evERdock.

    Science.gov (United States)

    Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

    2018-03-14

    To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

  2. Binding free energy analysis of protein-protein docking model structures by evERdock

    Science.gov (United States)

    Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

    2018-03-01

    To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

  3. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    Science.gov (United States)

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

  4. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  5. Identification of Putative Vero Cell Protein(s) that Bind Specifically to ...

    African Journals Online (AJOL)

    with a cellular membrane [3,4]. The initial binding of dengue virus to target cells is mediated by binding of the envelope protein to a specific and unidentified cell surface receptor(s) [5]. Viral interaction with its targets observed by electron microscopy has been already reported. [6]. The structure of the ectodomain of DEN-2E.

  6. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  7. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  8. Identification of two nuclear N-acetylglucosamine-binding proteins.

    Science.gov (United States)

    Felin, M; Doyennette-Moyne, M A; Hadj-Sahraoui, Y; Aubery, M; Hubert, J; Sève, A P

    1994-12-01

    Using neoglycoproteins, lectins that recognize different sugars, including N-acetylglucosamine residues, were previously detected in animal cell nuclei. We report herein the isolation of two N-acetylglucosamine-binding proteins from HL60 cell nuclei: i) a 22 kDa polypeptide (CBP22) with an isoelectric point of 4.5 was isolated for the first time and ii) a 70 kDa polypeptide with an isoelectric point of 7.8. This latter protein corresponds to the glucose-binding protein (CBP70) previously isolated, based on the following similarities: i) they have the same molecular mass, ii) they have the same isoelectric point, iii) they are recognized by antibodies raised against CBP70, and iv) both are lectins from the C group of Drickamer's classification. CBP70 appeared to recognize glucose and N-acetylglucosamine; however, its affinity for N-acetylglucosamine was found to be twice that for glucose. The presence in the nucleus of two nuclear N-acetylglucosamine-binding proteins and their potential ligands, such as O-N-acetylglucosamine glycoproteins, strongly argues for possible intranuclear glycoprotein-lectin interactions.

  9. Role of adipocyte lipid-binding protein (ALBP) and acyl-coA binding protein (ACBP) in PPAR-mediated transactivation

    DEFF Research Database (Denmark)

    Helledie, Torben; Jørgensen, Claus; Antonius, Marianne

    2002-01-01

    lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization...... appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands....

  10. Regulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-ARegulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-A

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

    recognised role for PAPP-A in ageing and in the development of age-related disease. PAPP-A is a secreted metalloproteinase that cleaves insulin-like growth factor binding proteins (IGFBPs). Ervinas Gaidamauskas studied the mechanism of IGF-modulated proteolysis of IGFBPs by PAPP-A and the structural......During his PhD studies, Ervinas Gaidamauskas researched the proteins pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2 in vitro. As suggested by its name, PAPP-A plays an important role in pregnancy and fetal development. Additionally, recent studies indicate a newly...... determinants for cleavage. Using small-angle X-ray scattering (SAXS), he also analysed the intermodular structural organisation of the C-terminal domain of PAPP-A involved in substrate binding. Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role...

  11. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  12. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Science.gov (United States)

    Kirjavainen, Vesa; Jarva, Hanna; Biedzka-Sarek, Marta; Blom, Anna M; Skurnik, Mikael; Meri, Seppo

    2008-08-29

    Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

  13. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

    Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

  14. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Guan-Lin [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Wu, Jing-Yiing [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Yeh, Chang-Ching [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Kuo, Cheng-Chin, E-mail: kuocc@nhri.org.tw [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2016-05-13

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

  15. Surface selective binding of nanoclay particles to polyampholyte protein chains.

    Science.gov (United States)

    Pawar, Nisha; Bohidar, H B

    2009-07-28

    Binding of nanoclay (Laponite) to gelatin-A and gelatin-B (both polyampholytes) molecules was investigated at room temperature (25 degrees C) both experimentally and theoretically. The stoichiometric binding ratio between gelatin and Laponite was found to be strongly dependent on the solution ionic strength. Large soluble complexes were formed at higher ionic strengths of the solution, a result supported by data obtained from light scattering, viscosity, and zeta potential measurements. The binding problem was theoretically modeled by choosing a suitable two-body screened Coulomb potential, U(R(+)) = (q(-)/2epsilon)[(Q(-)/R(-))e(-kR(-))-(Q(+)/R(+))e(-kR(+))], where the protein dipole has charges Q(+) and Q(-) that are located at distances R(+) and R(-) from the point Laponite charge q(-) and the dispersion liquid has dielectric constant (epsilon). U(R(+)) accounted for electrostatic interactions between a dipole (protein molecule) and an effective charge (Laponite particle) located at an angular position theta. Gelatin-A and Laponite association was facilitated by a strong attractive interaction potential that led to preferential binding of the biopolymer chains to negatively charged face of Laponite particles. In the case of gelatin-B selective surf ace patch binding dominated the process where the positively charged rim and negatively charged face of the particles were selectively bound to the oppositely charged segments of the biopolymer. The equilibrium separation (R(e)) between the protein and nanoclay particle revealed monovalent salt concentration dependence given by R(e) approximately [NaCl](alpha) where alpha = 0.6+/-0.2 for gelatin-A and alpha = 0.4+/-0.2 for gelatin-B systems. The equilibrium separations were approximately 30% less compared to the gelatin-A system implying preferential short-range ordering of the gelatin-B-nanoclay pair in the solvent.

  16. Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.

    Science.gov (United States)

    Chen, Eileen; Joseph, Simpson

    2015-07-01

    Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS). Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  17. Structural and binding studies of SAP-1 protein with heparin.

    Science.gov (United States)

    Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2015-03-01

    SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

  18. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    Energy Technology Data Exchange (ETDEWEB)

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  19. Boar seminal plasma proteins and their binding prperties. A review

    Czech Academy of Sciences Publication Activity Database

    Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 69, - (2004), s. 461-475 ISSN 0010-0765 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : seminal plasma proteins * binding properties * spermadhesins Subject RIV: CE - Biochemistry Impact factor: 1.062, year: 2004

  20. Studies of Fibronectin-Binding Proteins of Streptococcus equi

    OpenAIRE

    Lannergård, Jonas; Flock, Margareta; Johansson, Staffan; Flock, Jan-Ingmar; Guss, Bengt

    2005-01-01

    Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously ...

  1. RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

    Science.gov (United States)

    2010-06-01

    lysed in triple- detergent RIPA buffer with protease inhibitor cocktail (Roche, Pleasanton, CA). For nuclear and cytoplasmic fractionation, the NE-PER kit...Posttranscriptional regulation of IL-13 in T cells: role of the RNA-binding protein HuR. The Journal of allergy and clinical immunology 2008, 121(4):853-859...and western blot analysis. Western analysis was performed as described previously.12 For detection of VEGFα and TSP1 from tumors, triple- detergent

  2. Mannan-binding proteins from boar seminal plasma

    Czech Academy of Sciences Publication Activity Database

    Jelínková-Slavíčková, Petra; Liberda, J.; Maňásková, Pavla; Ryšlavá, H.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 62, 1-2 (2004), s. 167-182 ISSN 0165-0378. [Congress of the European Society for Reproductive & Developmental Immunology /4./. Rhodes, 04.06.2003-06.06.2003] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MŠk VS96141; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : boar seminal plasma proteins * mannan-binding proteins * oviductal epithelium Subject RIV: CE - Biochemistry Impact factor: 2.726, year: 2004

  3. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    International Nuclear Information System (INIS)

    Blackburn, W.D.; Tallant, E.A.; Wallace, R.W.

    1986-01-01

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca 2+ . One mechanism by which Ca 2+ may trigger neutrophil activation is through Ca 2+ /calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca 2+ /CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and 125 I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound 125 I-CaM in a Ca 2+ -dependent manner. One predominant region of 125 I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca 2+ -dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 μg/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca 2+ -dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca 2+ /calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase

  4. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  5. Binding properties and immunolocalization of a fatty acid-binding protein in Giardia lamblia.

    Science.gov (United States)

    Hassan, S M T; Maache, M; de la Guardia, R Díaz; Córdova, O M; García, J R Gil; Galiana, M; Acuña Castroviejo, D; Martins, M; Osuna, Antonio

    2005-04-01

    We describe here a fatty acid-binding protein (FABP) isolated and purified from the parasitic protozoon Giardia lamblia. The protein has a molecular mass of 8 kDa and an isoelectric point of 4.96. A Scatchard analysis of the data at equilibrium revealed a dissociation constant of 3.12 x 10(-8) M when the labeled oleic acid was displaced by a 10-fold greater concentration of unlabeled oleic acid. Testosterone, sodium desoxycholate, taurocholate, metronidazol, and alpha-tocopherol, together with butyric, arachidonic, palmitic, retinoic, and glycocholic acids, were also bound to the protein. Assays with polyclonal antibodies revealed that the protein is located in the ventral disk and also appears in the dorsal membrane, the cytoplasm, and in the vicinity of the lipid vacuoles.

  6. Fluctuations in Mass-Action Equilibrium of Protein Binding Networks

    Science.gov (United States)

    Yan, Koon-Kiu; Walker, Dylan; Maslov, Sergei

    2008-12-01

    We consider two types of fluctuations in the mass-action equilibrium in protein binding networks. The first type is driven by slow changes in total concentrations of interacting proteins. The second type (spontaneous) is caused by quickly decaying thermodynamic deviations away from equilibrium. We investigate the effects of network connectivity on fluctuations by comparing them to scenarios in which the interacting pair is isolated from the network and analytically derives bounds on fluctuations. Collective effects are shown to sometimes lead to large amplification of spontaneous fluctuations. The strength of both types of fluctuations is positively correlated with the complex connectivity and negatively correlated with complex concentration. Our general findings are illustrated using a curated network of protein interactions and multiprotein complexes in baker’s yeast, with empirical protein concentrations.

  7. Decreased protein binding of moxifloxacin in patients with sepsis?

    Directory of Open Access Journals (Sweden)

    Dorn, Christoph

    2017-02-01

    Full Text Available The mean (SD unbound fraction of moxifloxacin in plasma from patients with severe sepsis or septic shock was determined by ultrafiltration to 85.5±3.0% (range 81.9 and 91.6% indicating a decreased protein binding of moxifloxacin in this population compared with the value of 58–60% provided in the Summary of Product Characteristics. However, previous investigations neglected the influence of pH and temperature on the protein binding of moxifloxacin. Maintaining physiological conditions (pH 7.4, 37°C – as in the present study – the unbound fraction of moxifloxacin in plasma from healthy volunteers was 84%. In contrast, the unbound fraction of moxifloxacin was 77% at 4°C and 66–68% in unbuffered plasma or at pH 8.5 in fair agreement with previously published data. PK/PD parameters e.g. AUC/MIC or ratios between interstitial fluid and free plasma concentrations, which were obtained assuming a protein binding rate of moxifloxacin of 40% or more, should be revised.

  8. PRODIGY : a web server for predicting the binding affinity of protein-protein complexes

    NARCIS (Netherlands)

    Xue, Li; Garcia Lopes Maia Rodrigues, João; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-01-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given

  9. Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian

    2006-01-01

    Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present...

  10. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Science.gov (United States)

    Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

  11. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  12. The BRCT domain is a phospho-protein binding domain.

    Science.gov (United States)

    Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

    2003-10-24

    The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

  13. A conserved NAD+binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  14. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  15. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    NARCIS (Netherlands)

    Bosdriesz, E.; Magnúsdóttir, S.; Bruggeman, F.J.; Teusink, B.; Molenaar, D.

    2015-01-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is

  16. Activity of cefixime against Helicobacter pylori and affinities for the penicillin-binding proteins.

    Science.gov (United States)

    Ikeda, F; Yokota, Y; Mine, Y; Tatsuta, M

    1990-12-01

    Cefixime induced the formation of rounded cells from the spiral bacillary form of Helicobacter pylori at the MIC or less. Three main penicillin-binding proteins, called A, B and C, were separated from H. pylori. Cefixime had the strongest affinity to penicillin-binding protein B. The binding of cefixime to this protein may induce the formation of rounded H. pylori cells.

  17. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

  18. Collagen-binding proteins of Streptococcus mutans and related streptococci.

    Science.gov (United States)

    Avilés-Reyes, A; Miller, J H; Lemos, J A; Abranches, J

    2017-04-01

    The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  20. Folding energetics of ligand binding proteins. I. Theoretical model.

    Science.gov (United States)

    Rösgen, J; Hinz, H J

    2001-03-02

    Heat capacity curves as obtained from differential scanning calorimetry are an outstanding source for molecular information on protein folding and ligand-binding energetics. However, deconvolution of C(p) data of proteins in the presence of ligands can be compromised by indeterminacies concerning the correct choice of the statistical thermodynamic ensemble. By convent, the assumption of constant free ligand concentration has been used to derive formulae for the enthalpy. Unless the ligand occurs at large excess, this assumption is incorrect. Still the relevant ensemble is the grand canonical ensemble. We derive formulae for both constraints, constancy of total or free ligand concentration and illustrate the equations by application to the typical equilibrium Nx N + x D + x. It is demonstrated that as long as the thermodynamic properties of the ligand can be completely corrected for by performing a reference measurement, the grand canonical approach provides the proper and mathematically significantly simpler choice. We demonstrate on the two cases of sequential or independent ligand-binding the fact, that similar binding mechanisms result in different and distinguishable heat capacity equations. Finally, we propose adequate strategies for DSC experiments as well as for obtaining first estimates of the characteristic thermodynamic parameters, which can be used as starting values in a global fit of DSC data. Copyright 2001 Academic Press.

  1. Retinoic acid binding protein in normal and neopolastic rat prostate.

    Science.gov (United States)

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  2. The Collagen Binding Proteins of Streptococcus mutans and Related Streptococci

    Science.gov (United States)

    Avilés-Reyes, Alejandro; Miller, James H.; Lemos, José A.; Abranches, Jacqueline

    2016-01-01

    Summary The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms utilized by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. PMID:26991416

  3. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

  4. A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Sorensen, Boe S

    2012-01-01

    Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin......-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases......, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding...

  5. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    Science.gov (United States)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  6. The Movable Type Method Applied to Protein-Ligand Binding.

    Science.gov (United States)

    Zheng, Zheng; Ucisik, Melek N; Merz, Kenneth M

    2013-12-10

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term "movable type". Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the free

  7. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  8. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Science.gov (United States)

    Das, Shubhadip; Paul, Sandip

    2018-01-01

    The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS) cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  9. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  10. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  11. Transcriptional regulation of PP2A-A alpha is mediated by multiple factors including AP-2alpha, CREB, ETS-1, and SP-1.

    Directory of Open Access Journals (Sweden)

    He-Ge Chen

    2009-09-01

    Full Text Available Protein phosphatases-2A (PP-2A is a major serine/threonine phosphatase and accounts for more than 50% serine/threonine phosphatase activity in eukaryotes. The holoenzyme of PP-2A consists of the scaffold A subunit, the catalytic C subunit and the regulatory B subunit. The scaffold subunits, PP2A-A alpha/beta, provide a platform for both C and B subunits to bind, thus playing a crucial role in providing specific PP-2A activity. Mutation of the two genes encoding PP2A-A alpha/beta leads to carcinogenesis and likely other human diseases. Regulation of these genes by various factors, both extracellular and intracellular, remains largely unknown. In the present study, we have conducted functional dissection of the promoter of the mouse PP2A-A alpha gene. Our results demonstrate that the proximal promoter of the mouse PP2A-A alpha gene contains numerous cis-elements for the binding of CREB, ETS-1, AP-2 alpha, SP-1 besides the putative TFIIB binding site (BRE and the downstream promoter element (DPE. Gel mobility shifting assays revealed that CREB, ETS-1, AP-2 alpha, and SP-1 all bind to PP2A-A alpha gene promoter. In vitro mutagenesis and reporter gene activity assays reveal that while SP-1 displays negative regulation, CREB, ETS-1 and AP-2A alpha all positively regulate the promoter of the PP2A-A alpha gene. ChIP assays further confirm that all the above transcription factors participate the regulation of PP2A-A alpha gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-A alpha gene.

  12. Rbfox2 controls autoregulation in RNA-binding protein networks.

    Science.gov (United States)

    Jangi, Mohini; Boutz, Paul L; Paul, Prakriti; Sharp, Phillip A

    2014-03-15

    The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

  13. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

    OpenAIRE

    Swanson, M S; Dreyfuss, G

    1988-01-01

    Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. Thes...

  14. Comparison of two methods forecasting binding rate of plasma protein.

    Science.gov (United States)

    Hongjiu, Liu; Yanrong, Hu

    2014-01-01

    By introducing the descriptors calculated from the molecular structure, the binding rates of plasma protein (BRPP) with seventy diverse drugs are modeled by a quantitative structure-activity relationship (QSAR) technique. Two algorithms, heuristic algorithm (HA) and support vector machine (SVM), are used to establish linear and nonlinear models to forecast BRPP. Empirical analysis shows that there are good performances for HA and SVM with cross-validation correlation coefficients Rcv(2) of 0.80 and 0.83. Comparing HA with SVM, it was found that SVM has more stability and more robustness to forecast BRPP.

  15. Taurine promotes cognitive function in prenatally stressed juvenile rats via activating the Akt-CREB-PGC1α pathway.

    Science.gov (United States)

    Jia, Ning; Sun, Qinru; Su, Qian; Dang, Shaokang; Chen, Guomin

    2016-12-01

    Substantial evidence has shown that the oxidative damage to hippocampal neurons is associated with the cognitive impairment induced by adverse stimuli during gestation named prenatal stress (PS). Taurine, a conditionally essential amino acid, possesses multiple roles in the brain as a neuromodulator or antioxidant. In this study, to explore the roles of taurine in PS-induced learning and memory impairment, prenatal restraint stress was set up and Morris water maze (MWM) was employed for testing the cognitive function in the one-month-old rat offspring. The mitochondrial reactive oxygen species (ROS) level,mitochondrial membrane potential (MMP), ATP and cytochrome c oxidase (CcO) activity and apoptosis-related proteins in the hippocampus were detected. The activity of the Akt-cyclic AMP response element-binding protein (CREB)-peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) pathway in the hippocampus was measured. The results showed that high dosage of taurine administration in the early postnatal period attenuated impairment of spatial learning and memory induced by PS. Meanwhile, taurine administration diminished the increase in mitochondrial ROS, and recovered the reduction of MMP, ATP level and the activities of CcO, superoxide dismutase 2 (SOD2) and catalase induced by PS in the hippocampus. In addition, taurine administration recovered PS-suppressed SOD2 expression level. Taurine administration blocked PS-induced decrease in the ratio of Bcl-2/Bax and increase in the ratio of cleaved caspase-3/full-length caspase-3. Notably, taurine inhibited PS-decreased phosphorylation of Akt (pAkt) and phosphorylation of CREB (pCREB), which consequently enhanced the mRNA and protein levels of PGC1α. Taken together, these results suggest that high dosage of taurine administration during the early postnatal period can significantly improve the cognitive function in prenatally stressed juvenile rats via activating the Akt-CREB-PGC1α pathway. Therefore

  16. Thermostability and reversibility of silver nanoparticle-protein binding.

    Science.gov (United States)

    Wang, Bo; Seabrook, Shane A; Nedumpully-Govindan, Praveen; Chen, Pengyu; Yin, Hong; Waddington, Lynne; Epa, V Chandana; Winkler, David A; Kirby, Jason K; Ding, Feng; Ke, Pu Chun

    2015-01-21

    The interactions between nanoparticles (NPs) and proteins in living systems are a precursor to the formation of a NP-protein "corona" that underlies cellular and organism responses to nanomaterials. However, the thermodynamic properties and reversibility of NP-protein interactions have rarely been examined. Using an automated, high-throughput and temperature-controlled dynamic light scattering (DLS) technique we observed a distinct hysteresis in the hydrodynamic radius of branched polyethyleneimine (BPEI) coated-silver nanoparticles (bAgNPs) exposed to like-charged lysozyme during the processes of heating and cooling, in contrast to the irreversible interactions between bAgNPs and oppositely charged alpha lactalbumin (ALact). Our discrete molecular dynamics (DMD) simulations offered a new molecular insight into the differential structure, dynamics and thermodynamics of bAgNPs binding with the two protein homologs and further revealed the different roles of the capping agents of citrate and BPEI in NP-protein interactions. This study facilitates our understanding of the transformation of nanomaterials in living systems, whose implications range from the field study of nanotoxicology to nanomaterials synthesis, nanobiotechnology and nanomedicine.

  17. Roles of RNA-Binding Proteins in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2016-02-01

    Full Text Available Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR, and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP with low complexity domains, called intrinsically disordered proteins (IDPs, and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs in a poly(ADP-ribose (PAR-dependent manner (unpublished data. DNA-dependent PARP1 (poly-(ADP ribose polymerase 1 makes key contributions in the DNA damage response (DDR network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  18. Protein-protein binding before and after photo-modification of albumin

    Science.gov (United States)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  19. Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography.

    Science.gov (United States)

    Cabanne, Charlotte; Pezzini, Jérôme; Joucla, Gilles; Hocquellet, Agnès; Barbot, Caroline; Garbay, Bertrand; Santarelli, Xavier

    2009-05-15

    Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.

  20. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  1. The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

    DEFF Research Database (Denmark)

    Schrøder, Jacob Morville

    centrosomal MT array and abnormally long centriole-associated rootlet filaments. Cells lacking EB1 also had stumpy cilia and a disorganized centrosomal MT array, but rootlet filaments appeared normal. Further, live imaging revealed increased release frequency of MTs from the centrosome upon EB1 or EB3......EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends. EB1 plays a role in regulating MT dynamics, localizing other MT-associated proteins to the plus end, and in regulating interactions of MTs with the cell cortex, mitotic kinetochores and different......, are required for assembly of primary cilia in cultured human cells. The EB3 - siRNA ciliary phenotype could be rescued by GFP-EB1 expression, and GFP-EB3 over expression resulted in elongated cilia. Transmission electron microscopy (TEM) revealed that EB3-depleted cells possess stumpy cilia, a disorganized...

  2. Ice cream structure modification by ice-binding proteins.

    Science.gov (United States)

    Kaleda, Aleksei; Tsanev, Robert; Klesment, Tiina; Vilu, Raivo; Laos, Katrin

    2018-04-25

    Ice-binding proteins (IBPs), also known as antifreeze proteins, were added to ice cream to investigate their effect on structure and texture. Ice recrystallization inhibition was assessed in the ice cream mixes using a novel accelerated microscope assay and the ice cream microstructure was studied using an ice crystal dispersion method. It was found that adding recombinantly produced fish type III IBPs at a concentration 3 mg·L -1 made ice cream hard and crystalline with improved shape preservation during melting. Ice creams made with IBPs (both from winter rye, and type III IBP) had aggregates of ice crystals that entrapped pockets of the ice cream mixture in a rigid network. Larger individual ice crystals and no entrapment in control ice creams was observed. Based on these results a model of ice crystals aggregates formation in the presence of IBPs was proposed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Tongqiao Huoxue Decoction ameliorates learning and memory defects in rats with vascular dementia by up-regulating the Ca(2+)-CaMKII-CREB pathway.

    Science.gov (United States)

    Ge, Chao-Liang; Wang, Xin-Ming; Huang, Zhao-Gang; Xia, Quan; Wang, Ning; Xu, Du-Juan

    2015-11-01

    The present study was aimed at determining the effects of Tongqiao Huoxue Decoction (TQHXD) on the Ca(2+)-CaMKII-CREB pathway and the memory and learning capacities of rats with vascular dementia (VD). The rat VD model was established by using an improved bilateral carotid artery ligation method. The Morris water maze experiment was used to evaluate the ethology of the VD rats following treatments with TQHXD at 3.01, 6.02, and 12.04 g·kg(-1) per day for 31 days. At the end of experiment, the hippocampus were harvested and analyzed. Western blotting and RT-PCR were used to measure the expression levels of calmodulin-binding protein kinase II(CaMKII), protein kinase A(PKA), cAMP-response element binding protein(CREB), and three N-methyl-D-aspartic acid receptor subunits (NR1, NR2A, and NR2B). Our results revealed that TQHXD could alleviate the loss of learning abilities and increase the memory capacity (P < 0.05 and P < 0.01 vs the model group, respectively). The treatment with 6.02 and 12.04 g·kg(-1) of TQHXD significantly up-regulated the Ca(2+)-CaMKII-CREB pathway in the hippocampus. In conclusion, TQHXD showed therapeutic effects on a bilateral carotid artery ligation-induced vascular dementia model, through the up-regulation of calcium signalling pathways. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  4. Immobilized sialyloligo-macroligand and its protein binding specificity.

    Science.gov (United States)

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

    We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

  5. Comprehensive review and empirical analysis of hallmarks of DNA-, RNA- and protein-binding residues in protein chains.

    Science.gov (United States)

    Zhang, Jian; Ma, Zhiqiang; Kurgan, Lukasz

    2017-12-15

    Proteins interact with a variety of molecules including proteins and nucleic acids. We review a comprehensive collection of over 50 studies that analyze and/or predict these interactions. While majority of these studies address either solely protein-DNA or protein-RNA binding, only a few have a wider scope that covers both protein-protein and protein-nucleic acid binding. Our analysis reveals that binding residues are typically characterized with three hallmarks: relative solvent accessibility (RSA), evolutionary conservation and propensity of amino acids (AAs) for binding. Motivated by drawbacks of the prior studies, we perform a large-scale analysis to quantify and contrast the three hallmarks for residues that bind DNA-, RNA-, protein- and (for the first time) multi-ligand-binding residues that interact with DNA and proteins, and with RNA and proteins. Results generated on a well-annotated data set of over 23 000 proteins show that conservation of binding residues is higher for nucleic acid- than protein-binding residues. Multi-ligand-binding residues are more conserved and have higher RSA than single-ligand-binding residues. We empirically show that each hallmark discriminates between binding and nonbinding residues, even predicted RSA, and that combining them improves discriminatory power for each of the five types of interactions. Linear scoring functions that combine these hallmarks offer good predictive performance of residue-level propensity for binding and provide intuitive interpretation of predictions. Better understanding of these residue-level interactions will facilitate development of methods that accurately predict binding in the exponentially growing databases of protein sequences. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  7. Immunochemical characterization of the brain glutamate binding protein

    International Nuclear Information System (INIS)

    Roy, S.

    1986-01-01

    A glutamate binding protein (GBP) was purified from bovine and rat brain to near homogeneity. Polyclonal antibodies were raised against this protein. An enzyme-linked-immunosorbent-assay was used to quantify and determine the specificity of the antibody response. The antibodies were shown to strongly react with bovine brain GBP and the analogous protein from rat brain. The antibodies did not show any crossreactivity with the glutamate metabolizing enzymes, glutamate dehydrogenase, glutamine synthetase and glutamyl transpeptidase, however it crossreacted moderately with glutamate decarboxylase. The antibodies were also used to define the possible physiologic activity of GBP in synaptic membranes. The antibodies were shown: (i) to inhibit the excitatory amino-acid stimulation of thiocyanate (SCN)flux, (ii) had no effect on transport of L-Glutamic acid across the synaptic membrane, and (iii) had no effect on the depolarization-induced release of L-glutamate. When the anti-GBP antibodies were used to localize and quantify the GBP distribution in various subcellular fractions and in brain tissue samples, it was found that the hippocampus had the highest immunoreactivity followed by the cerebral cortex, cerebellar cortex and caudate-putamen. The distribution of immunoreactivity in the subcellular fraction were as follows: synaptic membranes > crude mitochondrial fraction > homogenate > myelin. In conclusion these studies suggest that: (a) the rat brain GBP and the bovine brain GBP are immunologically homologous protein, (b) there are no structural similarities between the GBP and the glutamate metabolizing enzymes with the exception of glutamate decarboxylase and (c) the subcellular and regional distribution of the GBP immunoreactivity followed a similar pattern as observed for L-[ 3 H]-binding

  8. Prediction of protein-protein binding site by using core interface residue and support vector machine

    Directory of Open Access Journals (Sweden)

    Sun Zhonghua

    2008-12-01

    Full Text Available Abstract Background The prediction of protein-protein binding site can provide structural annotation to the protein interaction data from proteomics studies. This is very important for the biological application of the protein interaction data that is increasing rapidly. Moreover, methods for predicting protein interaction sites can also provide crucial information for improving the speed and accuracy of protein docking methods. Results In this work, we describe a binding site prediction method by designing a new residue neighbour profile and by selecting only the core-interface residues for SVM training. The residue neighbour profile includes both the sequential and the spatial neighbour residues of an interface residue, which is a more complete description of the physical and chemical characteristics surrounding the interface residue. The concept of core interface is applied in selecting the interface residues for training the SVM models, which is shown to result in better discrimination between the core interface and other residues. The best SVM model trained was tested on a test set of 50 randomly selected proteins. The sensitivity, specificity, and MCC for the prediction of the core interface residues were 60.6%, 53.4%, and 0.243, respectively. Our prediction results on this test set were compared with other three binding site prediction methods and found to perform better. Furthermore, our method was tested on the 101 unbound proteins from the protein-protein interaction benchmark v2.0. The sensitivity, specificity, and MCC of this test were 57.5%, 32.5%, and 0.168, respectively. Conclusion By improving both the descriptions of the interface residues and their surrounding environment and the training strategy, better SVM models were obtained and shown to outperform previous methods. Our tests on the unbound protein structures suggest further improvement is possible.

  9. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  10. Methamphetamine addiction: involvement of CREB and neuroinflammatory signaling pathways.

    Science.gov (United States)

    Krasnova, Irina N; Justinova, Zuzana; Cadet, Jean Lud

    2016-05-01

    Addiction to psychostimulant methamphetamine (METH) remains a major public health problem in the world. Animal models that use METH self-administration incorporate many features of human drug-taking behavior and are very helpful in elucidating mechanisms underlying METH addiction. These models are also helping to decipher the neurobiological substrates of associated neuropsychiatric complications. This review summarizes our work on the influence of METH self-administration on dopamine systems, transcription and immune responses in the brain. We used the rat model of METH self-administration with extended access (15 h/day for eight consecutive days) to investigate the effects of voluntary METH intake on the markers of dopamine system integrity and changes in gene expression observed in the brain at 2 h-1 month after cessation of drug exposure. Extended access to METH self-administration caused changes in the rat brain that are consistent with clinical findings reported in neuroimaging and postmortem studies of human METH addicts. In addition, gene expression studies using striatal tissues from METH self-administering rats revealed increased expression of genes involved in cAMP response element binding protein (CREB) signaling pathway and in the activation of neuroinflammatory response in the brain. These data show an association of METH exposure with activation of neuroplastic and neuroinflammatory cascades in the brain. The neuroplastic changes may be involved in promoting METH addiction. Neuroinflammatory processes in the striatum may underlie cognitive deficits, depression, and parkinsonism reported in METH addicts. Therapeutic approaches that include suppression of neuroinflammation may be beneficial to addicted patients.

  11. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  12. Salicylate clearance, the resultant of protein binding and metabolism.

    Science.gov (United States)

    Furst, D E; Tozer, T N; Melmon, K L

    1979-09-01

    Steady-state plasma salicylate concentrations and protein binding were examined in 9 normal subjects to determine relationships among daily dose, total and unbound salicylate concentrations, and total and unbound clearances. Aspirin doses ranging from 0.66 to 4.0 mg/kg/hr were given to steady state. Free and total salicylate concentrations were measured with spectrophotometric, fluorimetric, and equilibrium dialysis techniques. Although unbound clearance decreased over the therapeutic range, total clearance was unchanged. The former is a consequence of saturable metabolism; the latter, of saturable plasma protein binding as well as saturable metabolism. The fraction unbound increased linearly with unbound concentration. Clearance determined at 1.8 mg/kg/hr was used to predict levels obtained at higher aspirin doses. Analysis of residuals was used to ascertain the accuracy of the prediction. The coefficient of variation from prediction among subjects was found to be +/- 14%. It is concluded that, in normal subjects, salicylate clearance changes relatively little over the therapeutic range because the increasing fraction unbound compensates for decreasing clearance of unbound drug.

  13. Tannin-binding salivary proteins in three captive rhinoceros species.

    Science.gov (United States)

    Clauss, Marcus; Gehrke, Janin; Hatt, Jean-Michel; Dierenfeld, Ellen S; Flach, Edmund J; Hermes, Robert; Castell, Johanna; Streich, W Juergen; Fickel, Joerns

    2005-01-01

    Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species.

  14. The telomere binding protein TRF2 induces chromatin compaction.

    Directory of Open Access Journals (Sweden)

    Asmaa M Baker

    2011-04-01

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  15. Vitis labruscana leaf extract ameliorates scopolamine-induced impairments with activation of Akt, ERK and CREB in mice.

    Science.gov (United States)

    Pariyar, Ramesh; Yoon, Chi-Su; Svay, Thida; Kim, Dae-Sung; Cho, Hyoung-Kwon; Kim, Sung Yeon; Oh, Hyuncheol; Kim, Youn-Chul; Kim, Jaehyo; Lee, Ho-Sub; Seo, Jungwon

    2017-12-01

    Grapes are among the most widely consumed plants and are used as a folk medicine. Vitis species have been traditionally used as anti-inflammatory, analgesic, and memory-enhancing agents, but, their biological activities of discarded grape leaves are not completely understood. We investigated the effects of alcoholic aqueous leaf extract of Vitis labruscana (LEVL) in a mouse model of memory impairment and tried to ascertain its mechanism. We also evaluated its effects in SH-SY5Y cells. LEVL (50, 100, and 150 mg/kg) was administered to ICR mice once daily for 7 days. Memory impairment was induced with intraperitoneal scopolamine injections (1 mg/kg) and measured with the Y-maze test and a passive avoidance task. LEVL-induced signaling was evaluated in SH-SY5Y cells and mouse hippocampi. We first identified quercetin-3-O-glucuronide as LEVL's major component. We then showed that LEVL promoted phosphorylation of Akt, extracellular regulated kinase (ERK), and cyclic AMP response element binding protein (CREB) and proliferation of SH-SY5Y cells. Oral LEVL administration (100 mg/kg) for 7 days significantly reversed scopolamine-induced reductions of spontaneous alternation in the Y-maze test and scopolamine-induced shortening of latency times in the passive avoidance task's retention trial. Consistent with the cell experiment results, LEVL restored scopolamine-decreased phosphorylation of Akt, ERK, and CREB and scopolamine-reduced expression of brain-derived neuroprotective factor expression in mouse hippocampi. Our results suggest that LEVL promotes phosphorylation of Akt, ERK, and CREB in the hippocampus and ameliorates scopolamine-induced memory impairment in mice. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show...... that depletion of ACBP in mammalian cells results in lethality, suggesting that ACBP is an essential protein....

  17. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  18. Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

    Science.gov (United States)

    Guo, Xiucai; Lin, Dongju; Li, Weiwei; Wang, Kai; Peng, Ying; Zheng, Jiang

    2016-03-21

    Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ.

  19. Metals and Neuronal Metal Binding Proteins Implicated in Alzheimer's Disease

    Science.gov (United States)

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent age-related dementia affecting millions of people worldwide. Its main pathological hallmark feature is the formation of insoluble protein deposits of amyloid-β and hyperphosphorylated tau protein into extracellular plaques and intracellular neurofibrillary tangles, respectively. Many of the mechanistic details of this process remain unknown, but a well-established consequence of protein aggregation is synapse dysfunction and neuronal loss in the AD brain. Different pathways including mitochondrial dysfunction, oxidative stress, inflammation, and metal metabolism have been suggested to be implicated in this process. In particular, a body of evidence suggests that neuronal metal ions such as copper, zinc, and iron play important roles in brain function in health and disease states and altered homeostasis and distribution as a common feature across different neurodegenerative diseases and aging. In this focused review, we overview neuronal proteins that are involved in AD and whose metal binding properties may underlie important biochemical and regulatory processes occurring in the brain during the AD pathophysiological process. PMID:26881049

  20. Isolation and Purification of Thiamine Binding Protein from Mung Bean

    Directory of Open Access Journals (Sweden)

    DWIRINI RETNO GUNARTI

    2013-03-01

    Full Text Available Thiamine has fundamental role in energy metabolism. The organs mostly sensitive to the lack of thiamine levels in the body are the nervous system and the heart. Thiamine deficiency causes symptoms of polyneuritis and cardiovascular diseases. Because of its importance in the metabolism of carbohydrates, we need to measure the levels of thiamine in the body fluids by using an easy and inexpensive way without compromising the sensitivity and selectivity. An option to it is thiamine measurement based on the principle of which is analogous to ELISA, in which a thiamine binding protein (TBP act by replacing antibodies. The presence of TBP in several seeds have been reported by previous researchers, but the presence of TBP in mung beans has not been studied. This study was aimed to isolate and purify TBP from mung bean. The protein was isolated from mung bean through salting out by ammonium sulphate of 40, 70, and 90% (w/v. TBP has a negative charge as shown by cellulose acetate electrophoresis. The result obtained after salting out by ammonium sulphate was further purified bymeans of DEAE-cellulose chromatography and affinity chromatography. In precipitation of 90% of salting out method, one peak protein was obtained by using affinity chromatography. The protein was analyzed by SDS PAGE electrophoresis. The result of SDS PAGE electrophoresis showed that TBP has a molecular weight of 72.63 kDa.

  1. Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit.

    Science.gov (United States)

    Herington, A C; Ymer, S; Roupas, P; Stevenson, J

    1986-04-11

    Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.

  2. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    International Nuclear Information System (INIS)

    Bojadzsieva, Milka; Kocsar, Laszlo; Kremmer, Tibor

    1985-01-01

    A micromethod was developed to determine the binding of anabolic streoids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline. (L.E.)

  3. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

  4. Identification of novel DNA binding proteins using DNA affinity chromatography-pulldown

    OpenAIRE

    Jutras, Brandon L; Verma, Ashutosh; Stevenson, Brian

    2012-01-01

    Methods are presented through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, then incubated with bacterial cytoplasmic extract. Washes with buffers containing non-specific DNA and low salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified...

  5. The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

    DEFF Research Database (Denmark)

    Jessen, U; Novitskaya, V; Pedersen, N

    2001-01-01

    form of the MAPK kinase, MEK2. Alternatively, PC12-E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c-Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c...

  6. Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness.

    Science.gov (United States)

    Conkright, Michael D; Guzmán, Ernesto; Flechner, Lawrence; Su, Andrew I; Hogenesch, John B; Montminy, Marc

    2003-04-01

    We have employed a hidden Markov model (HMM) based on known cAMP responsive elements to search for putative CREB target genes. The best scoring sites were positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments revealed a core promoter requirement for transcriptional induction via CREB; TATA-less promoters were unresponsive to cAMP compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescued cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration.

  7. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  8. Growth hormone receptor/binding protein: physiology and function.

    Science.gov (United States)

    Herington, A C; Ymer, S I; Stevenson, J L; Roupas, P

    1994-07-01

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular form(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR.

  9. Growth hormone receptor/binding protein: Physiology and function

    Energy Technology Data Exchange (ETDEWEB)

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P. [Royal Children`s Hospital, Melbourne (Australia)

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  10. ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

    Science.gov (United States)

    Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

  11. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  12. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  13. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    International Nuclear Information System (INIS)

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

    2007-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies

  14. STRUCTURAL CHARACTERIZATION OF THE RNA BINDING DOMAIN OF HUMAN STEM LOOP BINDING PROTEIN

    Directory of Open Access Journals (Sweden)

    Maruthi Kashyap

    2013-12-01

    Full Text Available A gene encoding the RNA binding domain (RBD of human stem loop binding protein (SLBP was cloned in pET 28a vector and over-expressed in E. coli codon plus cells. The over-expressed SLBP-RBD carried no tag and aggregated as inclusion bodies in the cell lysate. Inclusion bodies were semi-purified to >85% purity by establishing a method involving detergent washing and subsequently denatured in 8 M urea. Refolding of the denatured RBD was carried out by step dialysis in decreasing concentrations of urea and L-arginine. Refolded SLBP-RBD was analyzed using size exclusion chromatography that revealed its monomeric nature and folded state. Uniformly 15N and 15N,13C labeled SLBP-RBD was prepared at concentrations for solution NMR studies. Approximately, 60% of the sequence specific backbone resonance assignments have been achieved through standard triple resonance NMR experiments. Analyses of secondary chemical shifts reveal presence of a small helical secondary structural elements and large intrinsically disordered regions.

  15. Crystal structure of Methanococcus jannaschii TATA box-binding protein.

    Science.gov (United States)

    Adachi, Naruhiko; Senda, Miki; Natsume, Ryo; Senda, Toshiya; Horikoshi, Masami

    2008-11-01

    As the archaeal transcription system consists of a eukaryotic-type transcription apparatus and bacterial-type regulatory transcription factors, analyses of the molecular interface between the transcription apparatus and regulatory transcription factors are critical to reveal the evolutionary change of the transcription system. TATA box-binding protein (TBP), the central components of the transcription apparatus are classified into three groups: eukaryotic, archaeal-I and archaeal-II TBPs. Thus, comparative functional analysis of these three groups of TBP is important for the study of the evolution of the transcription system. Here, we present the first crystal structure of an archaeal-II TBP from Methanococcus jannaschii. The highly conserved and group-specific conserved surfaces of TBP bind to DNA and TFIIB/TFB, respectively. The phylogenetic trees of TBP and TFIIB/TFB revealed that they evolved in a coupled manner. The diversified surface of TBP is negatively charged in the archaeal-II TBP, which is completely different from the case of eukaryotic and archaeal-I TBPs, which are positively charged and biphasic, respectively. This difference is responsible for the diversification of the regulatory functions of TBP during evolution.

  16. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  17. Relative role of upstream regulators of Akt, ERK and CREB in NCAM- and FGF2-mediated signalling

    DEFF Research Database (Denmark)

    Ditlevsen, D.K.; Owczarek, S.; Berezin, V.

    2008-01-01

    with an NCAM ligand, the C3d peptide. NCAM-mediated ERK phosphorylation depended on activation of the fibroblast growth factor receptor (FGFR), Src-family kinases, MEK (MAP and ERK kinase) and G(0)/G(i)-proteins, whereas NCAM-mediated CREB phosphorylation depended on the activity of Src-family kinases and MEK...... for phosphorylation of ERK, Akt, and CREB. MEK was required for phosphorylation of ERK and CREB, but not Akt, whereas G(0)/G(i)-proteins were necessary for phosphorylation of Akt and CREB, and cGMP was necessary for Akt phosphorylation. We thus demonstrate that even though NCAM and FGF2 have many signalling features...

  18. SFS, a Novel Fibronectin-Binding Protein from Streptococcus equi, Inhibits the Binding between Fibronectin and Collagen

    Science.gov (United States)

    Lindmark, Hans; Guss, Bengt

    1999-01-01

    The obligate parasitic bacterium Streptococcus equi subsp. equi is the causative agent of strangles, a serious disease of the upper respiratory tract in horses. In this study we have, using shotgun phage display, cloned from S. equi subsp. equi and characterized a gene, called sfs, encoding a protein termed SFS, representing a new type of fibronectin (Fn)-binding protein. The sfs gene was found to be present in all 50 isolates of S. equi subsp. equi tested and in 41 of 48 S. equi subsp. zooepidemicus isolates tested. The sfs gene is down-regulated during growth in vitro compared to fnz, a previously characterized gene encoding an Fn-binding protein from S. equi subsp. zooepidemicus. Sequence comparisons revealed no similarities to previously characterized Fn-binding proteins, but high scores were obtained against collagen. Besides similarity due to the high content of glycine, serine, and proline residues present in both proteins, there was a nine-residue motif present both in collagen and in the Fn-binding domain of SFS. By searching the Oklahoma S. pyogenes database, we found that this motif is also present in a potential cell surface protein from S. pyogenes. Protein SFS was found to inhibit the binding between Fn and collagen in a concentration-dependent way. PMID:10225899

  19. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

    Science.gov (United States)

    Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

    1995-01-01

    The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

  20. Mannan-binding lectin in cerebrospinal fluid: a leptomeningeal protein

    Directory of Open Access Journals (Sweden)

    Reiber Hansotto

    2012-08-01

    Full Text Available Abstract Background Mannan-binding lectin (MBL, a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb, i.e., with CSF flow rate. Methods MBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction. Results MBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3 The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66% compared to the MBL concentrations in serum (CV = 146% indicate an independent source of MBL in CSF. 4 The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb. Conclusions MBL in CSF is

  1. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  2. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    DEFF Research Database (Denmark)

    Fattaey, A R; Helin, K; Dembski, M S

    1993-01-01

    The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one...

  3. Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions

    NARCIS (Netherlands)

    Julkowska, M.M.; Rankenberg, J.M.; Testerink, C.

    2013-01-01

    Protein-lipid interactions play an important role in cellular protein relocation, activation and signal transduction. The liposome-binding assay is a simple and inexpensive method to examine protein-lipid binding in vitro. The phospholipids used for liposome production are dried and hydrated.

  4. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  5. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    Science.gov (United States)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  6. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  7. Unified understanding of folding and binding mechanisms of globular and intrinsically disordered proteins.

    Science.gov (United States)

    Arai, Munehito

    2018-01-06

    Extensive experimental and theoretical studies have advanced our understanding of the mechanisms of folding and binding of globular proteins, and coupled folding and binding of intrinsically disordered proteins (IDPs). The forces responsible for conformational changes and binding are common in both proteins; however, these mechanisms have been separately discussed. Here, we attempt to integrate the mechanisms of coupled folding and binding of IDPs, folding of small and multi-subdomain proteins, folding of multimeric proteins, and ligand binding of globular proteins in terms of conformational selection and induced-fit mechanisms as well as the nucleation-condensation mechanism that is intermediate between them. Accumulating evidence has shown that both the rate of conformational change and apparent rate of binding between interacting elements can determine reaction mechanisms. Coupled folding and binding of IDPs occurs mainly by induced-fit because of the slow folding in the free form, while ligand binding of globular proteins occurs mainly by conformational selection because of rapid conformational change. Protein folding can be regarded as the binding of intramolecular segments accompanied by secondary structure formation. Multi-subdomain proteins fold mainly by the induced-fit (hydrophobic collapse) mechanism, as the connection of interacting segments enhances the binding (compaction) rate. Fewer hydrophobic residues in small proteins reduce the intramolecular binding rate, resulting in the nucleation-condensation mechanism. Thus, the folding and binding of globular proteins and IDPs obey the same general principle, suggesting that the coarse-grained, statistical mechanical model of protein folding is promising for a unified theoretical description of all mechanisms.

  8. Analysis list: CREB1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available CREB1 Blood,Digestive tract,Liver,Pluripotent stem cell,Prostate,Uterus + hg19 http...://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CREB1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CRE...B1.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CREB1.10.tsv http://dbarchive.b...iosciencedbc.jp/kyushu-u/hg19/colo/CREB1.Blood.tsv,http://dbarchive.biosciencedbc....jp/kyushu-u/hg19/colo/CREB1.Digestive_tract.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/CREB1.

  9. The effects of GH and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein D and vitamin D binding protein in Turner syndrome

    DEFF Research Database (Denmark)

    Gravholt, Claus Højbjerg; Leth-Larsen, Rikke; Lauridsen, Anna Lis

    2004-01-01

    function. In the present study we examined whether GH or hormone replacement therapy (HRT) in Turner syndrome (TS) influence the serum concentrations of MBL and two other proteins partaking in the innate immune defence, surfactant protein D (SP-D) and vitamin D binding protein (DBP). DESIGN: Study 1...

  10. Translation initiation mediated by nuclear cap-binding protein complex.

    Science.gov (United States)

    Ryu, Incheol; Kim, Yoon Ki

    2017-04-01

    In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

  11. Is vitamin D binding protein a novel predictor of labour?

    Directory of Open Access Journals (Sweden)

    Stella Liong

    Full Text Available Vitamin D binding protein (VDBP has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4-7, 8-14 and 15-28 days before labour (P<0.05. VDBP concentration was 4.3-fold significantly higher at 0-3 days compared to 15-28 days pre-labour (P<0.05. The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974. This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.

  12. QM/MM Molecular Dynamics Studies of Metal Binding Proteins

    Directory of Open Access Journals (Sweden)

    Pietro Vidossich

    2014-07-01

    Full Text Available Mixed quantum-classical (quantum mechanical/molecular mechanical (QM/MM simulations have strongly contributed to providing insights into the understanding of several structural and mechanistic aspects of biological molecules. They played a particularly important role in metal binding proteins, where the electronic effects of transition metals have to be explicitly taken into account for the correct representation of the underlying biochemical process. In this review, after a brief description of the basic concepts of the QM/MM method, we provide an overview of its capabilities using selected examples taken from our work. Specifically, we will focus on heme peroxidases, metallo-β-lactamases, α-synuclein and ligase ribozymes to show how this approach is capable of describing the catalytic and/or structural role played by transition (Fe, Zn or Cu and main group (Mg metals. Applications will reveal how metal ions influence the formation and reduction of high redox intermediates in catalytic cycles and enhance drug metabolism, amyloidogenic aggregate formation and nucleic acid synthesis. In turn, it will become manifest that the protein frame directs and modulates the properties and reactivity of the metal ions.

  13. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    Czech Academy of Sciences Publication Activity Database

    Siciliano, P.; He, X. L.; Woodcock, C.; Pickett, J. A.; Field, L. M.; Birkett, M. A.; Kalinová, Blanka; Gomulski, L. M.; Scolari, F.; Gasperi, G.; Malacrida, A. R.; Zhou, J. J.

    2014-01-01

    Roč. 48, May (2014), s. 51-62 ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

  14. 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...... defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP...

  15. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH 2 groups (CONH 2 -SWNT) exhibited very strong interactions between the CONH 2 -SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  16. Microarray analysis of androgen-regulated gene expression in testis: the use of the androgen-binding protein (ABP-transgenic mouse as a model

    Directory of Open Access Journals (Sweden)

    Grossman Gail

    2005-12-01

    Full Text Available Abstract Background Spermatogenesis is an androgen-dependent process, yet the molecular mechanisms of androgens' actions in testis are poorly understood. Transgenic mice overexpressing rat androgen-binding protein (ABP in their testes have reduced levels of intratesticular androgens and, as a result, show a progressive impairment of spermatogenesis. We used this model to characterize changes in global gene expression in testis in response to reduced bioavailability of androgens. Methods Total RNA was extracted from testes of 30-day old transgenic and wild-type control mice, converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays. Microarray results were confirmed by real-time reverse transcription polymerase chain reaction. Results Three-hundred-eighty-one genes (3.05% of all transcripts represented on the chips were up-regulated and 198 genes (1.59% were down-regulated by at least a factor of 2 in the androgen-deficient animals compared to controls. Genes encoding membrane proteins, intracellular signaling molecules, enzymes, proteins participating in the immune response, and those involved in cytoskeleton organization were significantly overrepresented in the up-regulated group. Among the down-regulated transcripts, those coding for extracellular proteins were overrepresented most dramatically, followed by those related to proteolysis, cell adhesion, immune response, and growth factor, cytokine, and ion channel activities. Transcripts with the greatest potential impact on cellular activities included several transcription factors, intracellular signal transducers, secreted signaling molecules and enzymes, and various cell surface molecules. Major nodes in the up-regulated network were IL-6, AGT, MYC, and A2M, those in the down-regulated network were IL-2, -4, and -10, MAPK8, SOCS1, and CREB1. Conclusion Microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional

  17. Conformational Dynamics of the Receptor Protein Galactose/Glucose Binding Protein

    Science.gov (United States)

    Messina, Troy; Talaga, David

    2006-03-01

    We have performed time-correlated single photon counting (TCSPC) anisotropy and Stokes Shift measurements on bulk solutions of galactose/glucose binding protein. Site-directed mutagenesis was used to provide a single cysteine amino acid near the sugar-binding center of the protein (glutamine 26 to cysteine -- Q26C). The cysteine was covalently labeled with the environmentally-sensitive fluorophore acrylodan, and a long-lived ruthenium complex was covalently attached to the N-terminus to provide a fluorescent reference. The TCSPC data were analyzed using global convolute-and-compare fitting routines over the entire glucose titration and temperature range to provide minimal reduced chi-squared values and the highest time resolution possible. Using a standard ligand-binding model, the resulting distributions show that the closed (ligand-bound) conformation exists even at zero glucose concentration. At 20^oC, the relative abundance of this conformation is as high as 40%. The temperature dependence of this conformational study will be discussed and related to the ligand-binding free energy surface.

  18. Metal binding is critical for the folding and function of laminin binding protein, Lmb of Streptococcus agalactiae.

    Directory of Open Access Journals (Sweden)

    Preethi Ragunathan

    Full Text Available Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.

  19. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  1. A robust assay to measure DNA topology-dependent protein binding affinity.

    Science.gov (United States)

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.

  2. Identification and quantification of calcium-binding proteins in squid axoplasm

    International Nuclear Information System (INIS)

    Krinks, M.H.; Klee, C.B.; Pant, H.C.; Gainer, H.

    1988-01-01

    The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. 45 Ca-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (Mr greater than 330 and 220 X 10(3] neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium-binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca 2+ /kg axoplasm. 125 I-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca 2+ /CaM protein kinase II subunits

  3. Interspecies In Vitro Evaluation of Stereoselective Protein Binding for 3,4-Methylenedioxymethamphetamine

    Directory of Open Access Journals (Sweden)

    Wan Raihana Wan Aasim

    2017-01-01

    Full Text Available Abuse of 3,4-methylenedioxymethamphetamine (MDMA is becoming more common worldwide. To date, there is no information available on stereoselectivity of MDMA protein binding in humans, rats, and mice. Since stereoselectivity plays an important role in MDMA’s pharmacokinetics and pharmacodynamics, in this study we investigated its stereoselectivity in protein binding. The stereoselective protein binding of rac-MDMA was investigated using two different concentrations (20 and 200 ng/mL in human plasma and mouse and rat sera using an ultrafiltration technique. No significant stereoselectivity in protein binding was observed in both human plasma and rat serum; however, a significant stereoselective binding (p<0.05 was observed in mouse serum. Since the protein binding of MDMA in mouse serum is considerably lower than in humans and rats, caution should be exercised when using mice for in vitro studies involving MDMA.

  4. CXCL4 is a novel nickel-binding protein and augments nickel allergy.

    Science.gov (United States)

    Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

    2017-08-01

    Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

  5. Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation.

    Science.gov (United States)

    Arvanitis, Demetrios A; Vafiadaki, Elizabeth; Papalouka, Vasiliki; Sanoudou, Despina

    2017-12-01

    Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca 2+ concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Assessing protein-ligand docking for the binding of organometallic compounds to proteins.

    Science.gov (United States)

    Ortega-Carrasco, Elisabeth; Lledós, Agusti; Maréchal, Jean-Didier

    2014-01-30

    Organometallic compounds are increasingly used as molecular scaffolds in drug development projects; their structural and electronic properties offering novel opportunities in protein-ligand complementarities. Interestingly, while protein-ligand dockings have long become a spearhead in computer assisted drug design, no benchmarking nor optimization have been done for their use with organometallic compounds. Pursuing our efforts to model metal mediated recognition processes, we herein present a systematic study of the capabilities of the program GOLD to predict the interactions of protein with organometallic compounds. The study focuses on inert systems for which no alteration of the first coordination sphere of the metal occurs upon binding. Several scaffolds are used as test systems with different docking schemes and scoring functions. We conclude that ChemScore is the most robust scoring function with ASP and ChemPLP providing with good results too and GoldScore slightly underperforming. This study shows that current state-of-the-art protein-ligand docking techniques are reliable for the docking of inert organometallic compounds binding to protein. Copyright © 2013 Wiley Periodicals, Inc.

  7. Cost Function Network-based Design of Protein-Protein Interactions: predicting changes in binding affinity.

    Science.gov (United States)

    Viricel, Clément; de Givry, Simon; Schiex, Thomas; Barbe, Sophie

    2018-02-20

    Accurate and economic methods to predict change in protein binding free energy upon mutation are imperative to accelerate the design of proteins for a wide range of applications. Free energy is defined by enthalpic and entropic contributions. Following the recent progresses of Artificial Intelligence-based algorithms for guaranteed NP-hard energy optimization and partition function computation, it becomes possible to quickly compute minimum energy conformations and to reliably estimate the entropic contribution of side-chains in the change of free energy of large protein interfaces. Using guaranteed Cost Function Network algorithms, Rosetta energy functions and Dunbrack's rotamer library, we developed and assessed EasyE and JayZ, two methods for binding affinity estimation that ignore or include conformational entropic contributions on a large benchmark of binding affinity experimental measures. If both approaches outperform most established tools, we observe that side-chain conformational entropy brings little or no improvement on most systems but becomes crucial in some rare cases. as open-source Python/C ++ code at sourcesup.renater.fr/projects/easy-jayz. thomas.schiex@inra.fr and sophie.barbe@insa-toulouse.fr. Supplementary data are available at Bioinformatics online.

  8. Efficient identification of phosphatidylserine-binding proteins by ORF phage display

    International Nuclear Information System (INIS)

    Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

    2009-01-01

    To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

  9. Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

    Science.gov (United States)

    Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

    2015-05-29

    Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

    Science.gov (United States)

    Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

    2017-07-18

    Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

  11. The interaction between anticoagulant protein S and complement regulatory C4b-binding protein (C4BP)

    NARCIS (Netherlands)

    van de Poel, R. H.; Meijers, J. C.; Bouma, B. N.

    2000-01-01

    An important mechanism of regulation of blood coagulation is the anticoagulant protein C pathway. In this pathway, the anticoagulant activity of activated protein C is increased by its cofactor protein S. The cofactor activity of protein S can be regulated by binding to complement regulatory

  12. Regulatory pathways for ATP-binding cassette transport proteins in kidney proximal tubules

    NARCIS (Netherlands)

    Masereeuw, R.; Russel, F.G.M.

    2012-01-01

    The ATP-binding cassette transport proteins (ABC transporters) represent important determinants of drug excretion. Protective or excretory tissues where these transporters mediate substrate efflux include the kidney proximal tubule. Regulation of the transport proteins in this tissue requires

  13. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  14. Dexamethasone-induced and estradiol-induced CREB activation and annexin 1 expression in CCRF-CEM lymphoblastic cells: evidence for the involvement of cAMP and p38 MAPK

    Directory of Open Access Journals (Sweden)

    M. Castro-caldas

    2003-01-01

    Full Text Available Aims: Annexin 1 (ANXA1, a member of the annexin family of calcium-binding and phospholipid-binding proteins, is a key mediator of the anti-inflammatory actions of steroid hormones. We have previously demonstrated that, in the human lymphoblastic CCRF-CEM cell line, both the synthetic glucocorticoid hormone, dexamethasone (Dex, and the estrogen hormone, 17β-estradiol (E2β, induce the synthesis of ANXA1, by a mechanism independent of the activation of their nuclear receptors. Recently, it was reported that the gene coding for ANXA1 contains a cAMP-responsive element (CRE. In this work, we investigated whether Dex and E2β were able to induce the activation of CRE binding proteins (CREB in the CCRF-CEM cells. Moreover, we studied the intracellular signalling pathways involved in CREB activation and ANXA1 synthesis in response to Dex and E2β; namely, the role of cAMP and the p38 mitogen-activated protein kinase (MAPK.

  15. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  16. (1)H, (13)C and (15)N resonance assignments of human FK506 binding protein 25.

    Science.gov (United States)

    Prakash, Ajit; Shin, Joon; Yoon, Ho Sup

    2015-04-01

    Human FKBP25, a nuclear protein, is a member of FK506 binding protein family (FKBP) and binds to immunosuppressive drugs such as FK506 and rapamycin. Human FKBP25 interacts with several nuclear proteins and regulates nuclear events. To understand the molecular basis of such interactions, we have performed NMR studies. Here, we report (1)H, (15)N and (13)C resonance assignments of the full-length human FKBP25 protein.

  17. Protein kinase A regulates AKAP250 (gravin) scaffold binding to the β2-adrenergic receptor

    OpenAIRE

    Tao, Jiangchuan; Wang, Hsien-yu; Malbon, Craig C.

    2003-01-01

    A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the β2-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor–scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold–receptor interactions. Gravin constructs lackin...

  18. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism.

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-04-30

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h ≈ 0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in a significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding.

  19. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-01-01

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

  20. Competitive protein binding analysis for thyroxine using Sephadex column (Tetralute)

    International Nuclear Information System (INIS)

    Miyai, Kiyoshi; Katayama, Yoshiaki; Sawazaki, Norio; Ishibashi, Kaichiro; Kawashima, Minoru.

    1975-01-01

    The method of competitive protein binding analysis of thyroxine (T 4 ) using Tetralute kit was evaluated. The net retention was decreased when the procedure of competition and separation was performed at a higher temperature but the final T 4 -I values were constant when the standard and test sera were treated identically. Coefficient of variation (C.V.) was 4% (within-assay) and 6% (between-assay) respectively. However, the T 4 -I values of pooled serum for quality control were slightly lower in earlier experiments in which correction factors (1.03--1.62 in 18 out of 21 assays) were necessary. T 4 -I values were determined by the Tetralute in 155 cases. They were as follows: 4.9+-0.8 μg/dl (euthyroid subjects), 6.4+-1.2 μg/dl (cord serum), 7.1+-1.1 μg/dl (pregnant women). 9.0+-3.6 μg/dl (trophoblastic disease), 13.3+-4.8 μg/dl (Graves' disease), 6.3+-1.6 μg/dl (Plummer's disease), 4 -I values determined by Tetralute and Res-O-Mat T 4 (r=0.96). Following oral administration of Telepaque the serum protein-bound iodine was markedly elevated, while the T 4 -I determined by Tetralute did not change. In vitro addition of diphenylhydantoin (500 μg/ml), salicylate (4 mg/ml) and phenobarbital (1 mg/ml) had no or little effect on T 4 determination by Tetralute. A high concentration of benzbromarone (0.1 mg/ml) caused a higher value of T 4 -I determined by Tetralute when added to a TBG solution but there was only a slight increase when it was added to serum. (auth.)

  1. Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis.

    Science.gov (United States)

    Ueki, Tatsuya; Kawakami, Norifumi; Toshishige, Masaaki; Matsuo, Koichi; Gekko, Kunihiko; Michibata, Hitoshi

    2009-10-01

    Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+. These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+.

  2. Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri.

    Science.gov (United States)

    Northey, Tom; Venthur, Herbert; De Biasio, Filomena; Chauviac, Francois-Xavier; Cole, Ambrose; Ribeiro, Karlos Antonio Lisboa; Grossi, Gerarda; Falabella, Patrizia; Field, Linda M; Keep, Nicholas H; Zhou, Jing-Jiang

    2016-04-22

    Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-β-farnesene but M. viciae uses a mixture of (E)-β-farnesene, (-)-α-pinene, β-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket.

  3. Glutamate affects the production of epoxyeicosanoids within the brain: The up-regulation of brain CYP2J through the MAPK-CREB signaling pathway.

    Science.gov (United States)

    Liu, Mingzhou; Zhu, Quanfei; Wu, Jinhua; Yu, Xuming; Hu, Mingbai; Xie, Xianfei; Yang, Zheqiong; Yang, Jing; Feng, Yu-Qi; Yue, Jiang

    2017-04-15

    Glutamate is the major excitatory neurotransmitter in the brain, and chronic glutamate excitotoxicity has been thought to be involved in numerous neurodegenerative diseases. We investigated the effects of glutamate at concentrations lower than the usual extrasynaptic concentrations on the production of epoxyeicosanoids mediated by brain CYP2J. Glutamate increased CYP2J2 mRNA levels in astrocytes in a dose-dependent manner, while an antagonist of the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) attenuated the glutamate-induced increases in CYP2J2 levels by glutamate. Glutamate increased the binding of cAMP response element-binding protein (CREB) with the CYP2J2 promoter, and the inhibition of the MAPK signaling pathway (ERK1/2, p38, and JNK) decreased the binding of CREB with the CYP2J2 promoter following the glutamate treatment. CREB activated the CYP2J2 promoter located at -1522 to -1317bp, and CREB overexpression significantly increased CYP2J2 mRNA levels. The CYP2J2 and mGlu5 mRNA levels were higher in the frontal cortex, hippocampus, cerebellum, and brainstem in adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7days of age. The data from the partial least-squares-discriminant analysis showed the epoxyeicosanoid profile of the hippocampus from the cerebellum, brain stem, and frontal cortex. The sum of the epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) was increased by 1.16-fold, 1.18-fold, and 1.19-fold in the frontal cortex, cerebellum, and brain stem, respectively, in rats treated with monosodium l-glutamate compared with the control group. The results suggest that brain CYP2J levels and CYP2J-mediated epoxyeicosanoid production can be regulated by extrasynaptic glutamate. The glutamate receptors expressed in astrocytes may mediate the regulation of drug-metabolizing enzymes and the metabolome of endogenous substances by glutamate. Copyright © 2017 Elsevier B.V. All rights

  4. Effects of single-stranded DNA binding proteins on primer extension by telomerase.

    Science.gov (United States)

    Cohen, Shlomit; Jacob, Eyal; Manor, Haim

    2004-08-12

    We present a biochemical analysis of the effects of three single-stranded DNA binding proteins on extension of oligonucleotide primers by the Tetrahymena telomerase. One of them, a human protein designated translin, which was shown to specifically bind the G-rich Tetrahymena and human telomeric repeats, slightly stimulated the primer extension reactions at molar ratios of translin/primer of primers, rather than by a direct interaction of this protein with telomerase. A second protein, the general human single-stranded DNA binding protein Replication Protein A (RPA), similarly affected the primer extension by telomerase, even though its mode of binding to DNA differs from that of translin. A third protein, the E. coli single-stranded DNA binding protein (SSB), whose binding to DNA is highly cooperative, caused more substantial stimulation and inhibition at the lower and the higher molar ratios of SSB/primer, respectively. Both telomere-specific and general single-stranded DNA binding proteins are found in living cells in telomeric complexes. Based on our data, we propose that these proteins may exert either stimulatory or inhibitory effects on intracellular telomerases, depending on their local concentrations. Copyright 2004 Elsevier B.V.

  5. Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck (Lepidoptera: Tortricidae.

    Directory of Open Access Journals (Sweden)

    Guangwei Li

    Full Text Available The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications.Two antennae-specific general OBPs (GOBPs of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2 for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1 exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition.Two rGmolGOBPs exhibit different binding characteristics for tested ligands. r

  6. Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

    Science.gov (United States)

    Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

    2016-01-01

    The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has

  7. Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

    Science.gov (United States)

    Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

    2016-01-01

    Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different

  8. Retinol binding protein 4, obesity, and insulin resistance in adolescents

    Directory of Open Access Journals (Sweden)

    Ronaldi Noor

    2017-02-01

    Full Text Available Background Obesity is a global problem. Even in poor and developing countries, obesity has reached alarming levels. In childhood, obesity may lead to insulin resistance. Retinol binding protein (RBP4, secreted primarily by liver and adipose tissues, was recently proposed as a link between obesity and insulin resistance. The role of RBP4 in pediatric obesity and its relationship with insulin resistance have not been well elucidated. Objective To compare RBP4 levels in obese and lean adolescents and to assess for a relationship between RBP4 levels and insulin resistance. Method This cross-sectional study was conducted in three senior high schools in Padang, West Sumatera, Indonesia. Subjects were adolescents aged 14-18 years, who were obese or normal weight (n=56. We measured subjects’ body mass index (BMI and serum RBP4 concentrations. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR index. Results Similar RBP4 levels were found in the obese and normoweight groups (P>0.05. Higher RBP4 levels were found in the insulin resistant compared to the non-insulin resistant group, but the difference was not significant (P > 0.05. Conclusion There is no significant difference in mean RBP4 levels in obese adolescents compared to normoweight adolescents. Nor are mean RBP4 levels significantly different between obese adolescents with and without insulin resistance.

  9. Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

    OpenAIRE

    Stirdivant, S M; Huber, H E; Patrick, D R; Defeo-Jones, D; McAvoy, E M; Garsky, V M; Oliff, A; Heimbrook, D C

    1992-01-01

    The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growt...

  10. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    Science.gov (United States)

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  11. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding...

  12. Vitamin D, vitamin D binding protein, lung function and structure in COPD

    DEFF Research Database (Denmark)

    Berg, Isaac; Hanson, Corrine; Sayles, Harlan

    2013-01-01

    Vitamin D and vitamin D binding protein (DBP) have been associated with COPD and FEV1. There are limited data regarding emphysema and vitamin D and DBP.......Vitamin D and vitamin D binding protein (DBP) have been associated with COPD and FEV1. There are limited data regarding emphysema and vitamin D and DBP....

  13. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each...

  14. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

  15. A small cellulose binding domain protein (CBD1) is highly variable in the nonbinding amino terminus

    Science.gov (United States)

    The small cellulose binding domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenophile Phytophthora infestans. Transgene expression of the protein in plants has also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. ...

  16. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    OpenAIRE

    Krug, Robert M.

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs).

  17. Effects of protein binding on the biodistribution of PEGylated PLGA nanoparticles post oral administration

    CSIR Research Space (South Africa)

    Semete, B

    2012-03-01

    Full Text Available of surface coating with various concentrations of polymeric surfactants (PEG and Pluronics F127) on the in vitro protein binding as well as the tissue biodistribution, post oral administration, of PLGA nanoparticles. The in vitro protein binding varied...

  18. Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

    Energy Technology Data Exchange (ETDEWEB)

    Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F. (NIH)

    2008-08-19

    Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.

  19. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    Science.gov (United States)

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  20. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose......-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted...

  1. Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

    Science.gov (United States)

    Stirdivant, S M; Huber, H E; Patrick, D R; Defeo-Jones, D; McAvoy, E M; Garsky, V M; Oliff, A; Heimbrook, D C

    1992-05-01

    The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological

  2. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119 ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D-fructose-binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  3. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Protein kinase Calpha contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities.

    Science.gov (United States)

    Slater, S J; Ho, C; Kelly, M B; Larkin, J D; Taddeo, F J; Yeager, M D; Stubbs, C D

    1996-03-01

    Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC alpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.

  5. The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange.

    Science.gov (United States)

    Qian, Yufeng; Johnson, Kenneth A

    2017-08-04

    The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB) 30 and (SSB) 60 , defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB) 60 and (SSB) 30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 10 9 m -1 s -1 ) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Cornelia Klein

    Full Text Available We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

  7. Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

    International Nuclear Information System (INIS)

    Langowski, J.; Benight, A.S.; Fujimoto, B.S.; Schurr, J.M.; Schomburg, U.

    1985-01-01

    The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D 0 ) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (D/sub app/) obtained from dynamic light scattering display the well-known increase with K 2 (K = scattering vector), leveling off toward a plateau value (D/sub plat/) at high K 2 . For both DNAs, the difference D/sub plat/ - D 0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed

  8. Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding

    Science.gov (United States)

    Armstrong, Anthony A.; Pardinas, Jose R.; Zheng, Songmao; Brosnan, Kerry; Emmell, Eva; Luo, Jeffrey; Chiu, Mark L.

    2017-01-01

    ABSTRACT The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies. PMID:28898162

  9. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  10. Γ-Ionizing radiation activated EGFR-p38/ERK-STAT3/CREB-1-EMT pathway for promotion of the migration/invasion of lung cancer cell and its inhibition by podophyllotoxin acetate

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Jeong Hyun; Um, Hong Duck; Park, Jong Kuk [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-05-15

    In this study, we sought to identify the intracellular machinery responsible for IR induced cancer invasion/migration. We report that IR activates the EGFR - p38/ERK - CREB-1/STAT3 pathway, which triggers EMT and increases invasion/migration of lung cancer. Moreover, we show that podophyllotoxin acetate (PA) inhibits IR-induced invasion/migration at least partly by blocking EGFR - p38/ERK - STAT3/ CREB-1signaling and thereby suppressing EMT. Our results revealed that IR increased the invasion/migration of A549 cells, and this effect was decreased by 10 nM PA treatment. PA also inhibited the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). The IR induced increases in invasion/migration were associated with the activation of EGFR-AKT, and PA inhibited this effect. P38 and p44/42 ERK were also involved in IR induced invasion/migration, and combined treatments with PA plus inhibitors of each MAPK synergistically blocked this invasion/migration. In terms of transcription factors (TFs), IR-induced increases in cyclic AMP response element-binding protein-1 (CREB-1) and signal transducer and activator of transcription 3 (STAT3) increased invasion/migration and EMT. PA also inhibited these transcription factors and then blocked IR-induced invasion/migration.