Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

Science.gov (United States)

Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

Directory of Open Access Journals (Sweden)

Bart J M Rooijakkers

Full Text Available Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

Distribution in rat tissues of modulator-binding protein of particulate nature

International Nuclear Information System (INIS)

Sobue, K.; Muramoto, Y.; Kakiuchi, S.; Yamazaki, R.

1979-01-01

Studies on Ca 2+ -activatable cyclic nucleotide phosphodiesterase led to the discovery of a protein modulator that is required for the activation of this enzyme by Ca 2+ . Later, this protein has been shown to cause the Ca 2+ -dependent activation of several enzymes that include phosphodiesterase, adenylate cyclase, a protein kinase from muscles, phosphorylase b kinase, actomyosin ATPase, membranous ATPase from erythrocytes and nerve synapses. Thus, modulator protein appears to be an intracellular mediator of actions of Ca 2+ . The present work shows the distribution of this particulate modulator-binding component in rat tissues. This paper also describes the labeling of modulator protein with tritium without deteriorating its biological activities and application of this 3 H-modulator protein to the determination of the Ca ++ dependent binding of modulator protein with membranous protein. This technique proves to be useful in studying enzymes or proteins whose functions are regulated by Ca ++ /modulator protein system. (Auth.)

Thermodynamic Characterization of New Positive Allosteric Modulators Binding to the Glutamate Receptor A2 Ligand-Binding Domain

DEFF Research Database (Denmark)

Nørholm, Ann-Beth; Francotte, Pierre; Goffin, Eric

2014-01-01

, and 5a (5-F) and 5b (6-F) are entropy driven. For 5d (8-F), both quantities were equal in size. Thermodynamic integration (TI) and one-step perturbation (OSP) were used to calculate the relative binding affinity of the modulators. The OSP calculations had a higher predictive power than those from TI......,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven......, and combined with the shorter total simulation time, we found the OSP method to be more effective for this setup. Furthermore, from the molecular dynamics simulations, we extracted the enthalpies and entropies, and along with the ITC data, this suggested that the differences in binding free energies...

Expression of an expansin carbohydrate-binding module affects ...

African Journals Online (AJOL)

Expansins are believed to be involved in disrupting the non-covalent adhesion of cellulose to matrix polysaccharides, thereby promoting wall creep. We have targeted a putative potato expansin (EXPA) carbohydrate-binding module (CBM) to the cell walls of tobacco plants. Histological examinations and electron ...

Monitoring non-proliferation of weapons of mass destruction: Can regional CBMs play a role?

International Nuclear Information System (INIS)

Sood, R.

1994-01-01

The experience of regional Confidence Building Measures (CBM), has only limited applicability for tackling proliferation of weapons of mass destruction. Where the international norm has developed as in the case of biological and chemical weapons, through international disarmament treaties, regional initiatives can strengthen this norm. Where a norm is less well-founded, regional initiatives are not likely to succeed. Specifically, with regard to nuclear weapons, consensus on negotiations for a comprehensive test ban treaty and a convention for prohibition of production of fissile materials for nuclear weapons purposes and explosive devices is a positive development. Successful conclusion of these universal and verifiable treaties will go a long way to strengthening the international norm against proliferation. Two other measures are critical - a development of a non-use assurance and commencement of multilateral nuclear disarmament negotiations among all five nuclear weapon States. If the international community witnesses improvement in these areas, regional negotiations will be stimulated. Therefore, the primary focus should be on developing an international norm to eliminate weapons of mass destruction. Regional efforts will take their cue from these international norms and would result in CBMs that are consistent with the international norm

Decorin binds myostatin and modulates its activity to muscle cells

International Nuclear Information System (INIS)

Miura, Takayuki; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito; Hennebry, Alex; Berry, Carole J.; Sharma, Mridula; Kambadur, Ravi; Nishimura, Takanori

2006-01-01

Myostatin, a member of TGF-β superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-β and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn 2+ greater than 10 μM, but not in the absence of Zn 2+ . Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K D ) of 2.02 x 10 -8 M and 9.36 x 10 -9 M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM

Membrane proteins bind lipids selectively to modulate their structure and function.

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Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

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Badino, Silke F; Christensen, Stefan J; Kari, Jeppe; Windahl, Michael S; Hvidt, Søren; Borch, Kim; Westh, Peter

2017-08-01

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 10 3 -10 4 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module.

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Matthews, Peter R; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E

2010-10-01

Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.

A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module

Science.gov (United States)

Matthews, Peter R.; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E.

2010-01-01

Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5′ and 3′ rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed. PMID:20702566

Allosteric enhancers, allosteric agonists and ago-allosteric modulators: where do they bind and how do they act?

DEFF Research Database (Denmark)

Schwartz, Thue W; Holst, Birgitte

2007-01-01

Many small-molecule agonists also display allosteric properties. Such ago-allosteric modulators act as co-agonists, providing additive efficacy--instead of partial antagonism--and they can affect--and often improve--the potency of the endogenous agonist. Surprisingly, the apparent binding sites...... different binding modes. In another, dimeric, receptor scenario, the endogenous agonist binds to one protomer while the ago-allosteric modulator binds to the other, 'allosteric' protomer. It is suggested that testing for ago-allosteric properties should be an integral part of the agonist drug discovery...... process because a compound that acts with--rather than against--the endogenous agonist could be an optimal agonist drug....

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

Science.gov (United States)

Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

2011-05-01

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

A phylogenetic study of SPBP and RAI1: evolutionary conservation of chromatin binding modules.

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Sagar Darvekar

Full Text Available Our genome is assembled into and array of highly dynamic nucleosome structures allowing spatial and temporal access to DNA. The nucleosomes are subject to a wide array of post-translational modifications, altering the DNA-histone interaction and serving as docking sites for proteins exhibiting effector or "reader" modules. The nuclear proteins SPBP and RAI1 are composed of several putative "reader" modules which may have ability to recognise a set of histone modification marks. Here we have performed a phylogenetic study of their putative reader modules, the C-terminal ePHD/ADD like domain, a novel nucleosome binding region and an AT-hook motif. Interactions studies in vitro and in yeast cells suggested that despite the extraordinary long loop region in their ePHD/ADD-like chromatin binding domains, the C-terminal region of both proteins seem to adopt a cross-braced topology of zinc finger interactions similar to other structurally determined ePHD/ADD structures. Both their ePHD/ADD-like domain and their novel nucleosome binding domain are highly conserved in vertebrate evolution, and construction of a phylogenetic tree displayed two well supported clusters representing SPBP and RAI1, respectively. Their genome and domain organisation suggest that SPBP and RAI1 have occurred from a gene duplication event. The phylogenetic tree suggests that this duplication has happened early in vertebrate evolution, since only one gene was identified in insects and lancelet. Finally, experimental data confirm that the conserved novel nucleosome binding region of RAI1 has the ability to bind the nucleosome core and histones. However, an adjacent conserved AT-hook motif as identified in SPBP is not present in RAI1, and deletion of the novel nucleosome binding region of RAI1 did not significantly affect its nuclear localisation.

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.

Monovalent cation and amiloride analog modulation of adrenergic ligand binding to the unglycosylated alpha 2B-adrenergic receptor subtype

International Nuclear Information System (INIS)

Wilson, A.L.; Seibert, K.; Brandon, S.; Cragoe, E.J. Jr.; Limbird, L.E.

1991-01-01

The unglycosylated alpha 2B subtype of the alpha 2-adrenergic receptor found in NG-108-15 cells possesses allosteric regulation of adrenergic ligand binding by monovalent cations and 5-amino-substituted amiloride analogs. These findings demonstrate that allosteric modulation of adrenergic ligand binding is not a property unique to the alpha 2A subtype. The observation that amiloride analogs as well as monovalent cations can modulate adrenergic ligand binding to the nonglycosylated alpha 2B subtype indicates that charge shielding due to carbohydrate moieties does not play a role in this allosteric modulation but, rather, these regulatory effects result from interactions of cations and amiloride analogs with the protein moiety of the receptor. Furthermore, the observation that both alpha 2A and alpha 2B receptor subtypes are modulated by amiloride analogs suggests that structural domains that are conserved between the two are likely to be involved in this allosteric modulation

Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

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Caitlin Siobhan Byrt

2012-11-01

Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

Science.gov (United States)

Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

2016-04-07

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose.

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Zhuolin Yi

Full Text Available During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A, which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9 module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c and TM2 (GH48 with three CBM3 modules synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other

Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose.

Science.gov (United States)

Yi, Zhuolin; Su, Xiaoyun; Revindran, Vanessa; Mackie, Roderick I; Cann, Isaac

2013-01-01

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases

Microscopic Analysis of Corn Fiber Using Corn Starch- and Cellulose-Specific Molecular Probes

Energy Technology Data Exchange (ETDEWEB)

Porter, S. E.; Donohoe, B. S.; Beery, K. E.; Xu, Q.; Ding, S.-Y.; Vinzant, T. B.; Abbas, C. A.; Himmel, M. E.

2007-09-01

Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.

Cellulose is not just cellulose

DEFF Research Database (Denmark)

Hidayat, Budi Juliman; Felby, Claus; Johansen, Katja Salomon

2012-01-01

are not regions where free cellulose ends are more abundant than in the bulk cell wall. In more severe cases cracks between fibrils form at dislocations and it is possible that the increased accessibility that these cracks give is the reason why hydrolysis of cellulose starts at these locations. If acid...... or enzymatic hydrolysis of plant cell walls is carried out simultaneously with the application of shear stress, plant cells such as fibers or tracheids break at their dislocations. At present it is not known whether specific carbohydrate binding modules (CBMs) and/or cellulases preferentially access cellulose...

Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

Science.gov (United States)

Ahmed, Ahmed H; Oswald, Robert E

2010-03-11

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

Module structure of interphotoreceptor retinoid-binding protein (IRBP may provide bases for its complex role in the visual cycle – structure/function study of Xenopus IRBP

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Ghosh Debashis

2007-08-01

Full Text Available Abstract Background Interphotoreceptor retinoid-binding protein's (IRBP remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. Results The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each ~300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 μM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites

Does protein binding modulate the effect of angiotensin II receptor antagonists?

Directory of Open Access Journals (Sweden)

Marc P Maillard

2001-03-01

Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

The carbohydrate-binding module family 20-diversity, structure, and function

DEFF Research Database (Denmark)

Christiansen, Camilla; Abou Hachem, Maher; Janecek, S.

2009-01-01

, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily......Starch-active enzymes often possess starch-binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate-binding module (CBM) families, and CBM20 is the earliest-assigned and best characterized family. High...... diversity characterizes CBM20s, which occur in starch-active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch-phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases...

Residues in the 5th module of the low-density lipoprotein receptor that bind apoE and apoB-100

International Nuclear Information System (INIS)

Kroon, P.A.; Zhang, H.-Y.; Smith, R.

2000-01-01

Full text: The low-density lipoprotein receptor (LDLR) binds and removes cholesterol-rich lipoproteins from the circulation. Its ligand-binding (LB) domain consists of seven cysteine-rich LB modules that bind apoB-100 and apoE. These modules fold into well-defined structures with three disulfide bonds, in the presence of Ca 2+ . The 5th module (LB5) is unique in that it is required to bind both apoB-100 and apoE. The aim of the current study was to map residues in human LB5 that are required for ligand binding. This was done by alanine mutagenesis of a series of residues that are conserved in human, mouse, rat and rabbit LB5 (E9, S14, E16, H19, S21, K31, and K33), but not in the other six modules. E37 (R37 in the rabbit) was included, since it has been previously hypothesized to play a role in binding. The variant LB5 modules were first produced as recombinant peptides, and subjected to oxidative folding to determine whether the mutations interfered with Ca 2+ '-dependent folding. Only the S14A and E16A mutations interfered significantly with folding, suggesting that S14 and E16 are required for the structural framework of LB5 and that their substitution in the LDLR may interfere with its folding. The native LDLR and E9A, H19A, S21A, K31A, K33A and E37A LDLRs were expressed in LDLR negative IdlA-7 CHO cells. Labeling with 125 I-lgG-C7 showed that all receptors were expressed on the cell surface. Binding of Dil-labeled LDL (Dil-LDL) and Dil-labeled DMPC, complexed with the N-terminal receptor-binding domain of apoE3 (Dil-E3), at 4 deg C, was used to assess receptor binding. Binding of Dil-E3 (0.1 μ/ml) to the H19A, S21A, K31A, K33A and E37A LDLRs was 65-92% of binding to the native LDLR. In contrast, the E9A LDLR only bound 3% of that of the native LDLR. The binding of Dil-LDL (0.5 Ag/ml) to the E9A LDLR was 23% of that of the native LDLR, while binding to the remaining variant LDLRs ranged from 44-70% of what of the native LDLR. We conclude that (i) E9 of LB5

Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

Directory of Open Access Journals (Sweden)

Eva Polanská

Full Text Available HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

Functional and modular analyses of diverse endoglucanases from Ruminococcus albus 8, a specialist plant cell wall degrading bacterium.

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Iakiviak, Michael; Devendran, Saravanan; Skorupski, Anna; Moon, Young Hwan; Mackie, Roderick I; Cann, Isaac

2016-07-21

Ruminococcus albus 8 is a specialist plant cell wall degrading ruminal bacterium capable of utilizing hemicellulose and cellulose. Cellulose degradation requires a suite of enzymes including endoglucanases, exoglucanases, and β-glucosidases. The enzymes employed by R. albus 8 in degrading cellulose are yet to be completely elucidated. Through bioinformatic analysis of a draft genome sequence of R. albus 8, seventeen putatively cellulolytic genes were identified. The genes were heterologously expressed in E. coli, and purified to near homogeneity. On biochemical analysis with cellulosic substrates, seven of the gene products (Ra0185, Ra0259, Ra0325, Ra0903, Ra1831, Ra2461, and Ra2535) were identified as endoglucanases, releasing predominantly cellobiose and cellotriose. Each of the R. albus 8 endoglucanases, except for Ra0259 and Ra0325, bound to the model crystalline cellulose Avicel, confirming functional carbohydrate binding modules (CBMs). The polypeptides for Ra1831 and Ra2535 were found to contain distantly related homologs of CBM65. Mutational analysis of residues within the CBM65 of Ra1831 identified key residues required for binding. Phylogenetic analysis of the endoglucanases revealed three distinct subfamilies of glycoside hydrolase family 5 (GH5). Our results demonstrate that this fibrolytic bacterium uses diverse GH5 catalytic domains appended with different CBMs, including novel forms of CBM65, to degrade cellulose.

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

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Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

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Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

2012-01-01

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

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Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

2010-12-01

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data*

Science.gov (United States)

Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

2010-01-01

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/. PMID:20733106

Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

Science.gov (United States)

Ahmed, Ahmed H.; Oswald, Robert E.

2010-01-01

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

Science.gov (United States)

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

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Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

Science.gov (United States)

Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

2016-09-01

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

Science.gov (United States)

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Overlapping binding site for the endogenous agonist, small-molecule agonists, and ago-allosteric modulators on the ghrelin receptor

DEFF Research Database (Denmark)

Holst, Birgitte; Frimurer, Thomas M; Mokrosinski, Jacek

2008-01-01

A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone......, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common....... It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists--including ago-allosteric modulators--and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor....

Growth modulation effects of CBM2a under the control of AtEXP4 and CaMV35S promoters in Arabidopsis thaliana, Nicotiana tabacum and Eucalyptus camaldulensis.

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Keadtidumrongkul, Pornthep; Suttangkakul, Anongpat; Pinmanee, Phitsanu; Pattana, Kanokwan; Kittiwongwattana, Chokchai; Apisitwanich, Somsak; Vuttipongchaikij, Supachai

2017-08-01

The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.

Secbase: database module to retrieve secondary structure elements with ligand binding motifs.

Science.gov (United States)

Koch, Oliver; Cole, Jason; Block, Peter; Klebe, Gerhard

2009-10-01

Secbase is presented as a novel extension module of Relibase. It integrates the information about secondary structure elements into the retrieval facilities of Relibase. The data are accessible via the extended Relibase user interface, and integrated retrieval queries can be addressed using an extended version of Reliscript. The primary information about alpha-helices and beta-sheets is used as provided by the PDB. Furthermore, a uniform classification of all turn families, based on recent clustering methods, and a new helix assignment that is based on this turn classification has been included. Algorithms to analyze the geometric features of helices and beta-strands were also implemented. To demonstrate the performance of the Secbase implementation, some application examples are given. They provide new insights into the involvement of secondary structure elements in ligand binding. A survey of water molecules detected next to the N-terminus of helices is analyzed to show their involvement in ligand binding. Additionally, the parallel oriented NH groups at the alpha-helix N-termini provide special binding motifs to bind particular ligand functional groups with two adjacent oxygen atoms, e.g., as found in negatively charged carboxylate or phosphate groups, respectively. The present study also shows that the specific structure of the first turn of alpha-helices provides a suitable explanation for stabilizing charged structures. The magnitude of the overall helix macrodipole seems to have no or only a minor influence on binding. Furthermore, an overview of the involvement of secondary structure elements with the recognition of some important endogenous ligands such as cofactors shows some distinct preference for particular binding motifs and amino acids.

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

Science.gov (United States)

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative analyses of two thermophilic enzymes exhibiting both beta-1,4 mannosidic and beta-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus.

Science.gov (United States)

Han, Yejun; Dodd, Dylan; Hespen, Charles W; Ohene-Adjei, Samuel; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

2010-08-01

The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

Science.gov (United States)

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii*

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Das, Subha Narayan; Wagenknecht, Martin; Nareddy, Pavan Kumar; Bhuvanachandra, Bhoopal; Niddana, Ramana; Balamurugan, Rengarajan; Swamy, Musti J.; Moerschbacher, Bruno M.; Podile, Appa Rao

2016-01-01

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici. Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction. PMID:27405759

C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

Science.gov (United States)

Kushwaha, Ambuj K; Grove, Anne

2013-01-24

Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis.

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Lung, Shiu-Cheung; Liao, Pan; Yeung, Edward C; Hsiao, An-Shan; Xue, Yan; Chye, Mee-Len

2017-07-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis ( Arabidopsis thaliana ) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1 Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 ( GL2 ), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1 , smo1-1 , and ACBP1 +/- smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1 +/- smo1-1 Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. © 2017 American Society of Plant Biologists. All Rights Reserved.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

Science.gov (United States)

Bohl, Casey E; Wu, Zengru; Chen, Jiyun; Mohler, Michael L; Yang, Jun; Hwang, Dong Jin; Mustafa, Suni; Miller, Duane D; Bell, Charles E; Dalton, James T

2008-10-15

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

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

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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.

A starch-binding domain identified in α-amylase (AmyP) represents a new family of carbohydrate-binding modules that contribute to enzymatic hydrolysis of soluble starch.

Science.gov (United States)

Peng, Hui; Zheng, Yunyun; Chen, Maojiao; Wang, Ying; Xiao, Yazhong; Gao, Yi

2014-04-02

A novel starch-binding domain (SBD) that represents a new carbohydrate-binding module family (CBM69) was identified in the α-amylase (AmyP) of the recently established alpha-amylase subfamily GH13_37. The SBD and its homologues come mostly from marine bacteria, and phylogenetic analysis indicates that they are closely related to the CBM20 and CBM48 families. The SBD exhibited a binding preference toward raw rice starch, but the truncated mutant (AmyPΔSBD) still retained similar substrate preference. Kinetic analyses revealed that the SBD plays an important role in soluble starch hydrolysis because different catalytic efficiencies have been observed in AmyP and the AmyPΔSBD. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

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Kang, Hyeran; Bradley, Michael J.; McCullough, Brannon R.; Pierre, Anaëlle; Grintsevich, Elena E.; Reisler, Emil; De La Cruz, Enrique M.

2012-01-01

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments. PMID:23027950

pH Modulates the Binding of EGR1 Transcription Factor to DNA

Science.gov (United States)

Mikles, David C.; Bhat, Vikas; Schuchardt, Brett J.; Deegan, Brian J.; Seldeen, Kenneth L.; McDonald, Caleb B.; Farooq, Amjad

2013-01-01

EGR1 transcription factor orchestrates a plethora of signaling cascades involved in cellular homeostasis and its down-regulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to non-ionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, H382 is predominantly conserved across other members of EGR1 family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating protein-DNA interactions central to this family of transcription factors. Collectively, our findings uncover an unexpected but a key step in the molecular recognition of EGR1 family of transcription factors and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

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Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

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Lisa M Maurer

Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

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Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines.

NARCIS (Netherlands)

Dube, N.; Delvin, E.; Yotov, W.; Garofalo, C.; Bendayan, M.; Veerkamp, J.H.; Levy, E.

2001-01-01

Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic

Bioinformatics Identification of Modules of Transcription Factor Binding Sites in Alzheimer's Disease-Related Genes by In Silico Promoter Analysis and Microarrays

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Regina Augustin

2011-01-01

Full Text Available The molecular mechanisms and genetic risk factors underlying Alzheimer's disease (AD pathogenesis are only partly understood. To identify new factors, which may contribute to AD, different approaches are taken including proteomics, genetics, and functional genomics. Here, we used a bioinformatics approach and found that distinct AD-related genes share modules of transcription factor binding sites, suggesting a transcriptional coregulation. To detect additional coregulated genes, which may potentially contribute to AD, we established a new bioinformatics workflow with known multivariate methods like support vector machines, biclustering, and predicted transcription factor binding site modules by using in silico analysis and over 400 expression arrays from human and mouse. Two significant modules are composed of three transcription factor families: CTCF, SP1F, and EGRF/ZBPF, which are conserved between human and mouse APP promoter sequences. The specific combination of in silico promoter and multivariate analysis can identify regulation mechanisms of genes involved in multifactorial diseases.

Modulation of [3H]-glutamate binding by serotonin in the rat hippocampus: An autoradiographic study

International Nuclear Information System (INIS)

Mennini, T.; Miari, A.

1991-01-01

Serotonin (5-HT) added in vitro increased [ 3 H]-glutamate specific binding in the rat hippocampus, reaching statistical significance in layers rich in N-Methyl-D-Aspartate sensitive glutamate receptors. This effect was explained by a significant increase in the apparent affinity of [ 3 H]-glutamate when 5-HT is added in vitro. Two days after lesion of serotonergic afferents to the hippocampus with 5,7- Dihydroxytryptamine [ 3 H]-glutamate binding was significantly decreased in the CA3 region and stratum lacunosum moleculare of the hippocampus, this reduction being reversed by in vitro addition of 10 μM 5-HT. The decrease observed is due to a significant reduction of quisqualate-insensitive (radiatum CA3) and kainate receptors (strata oriens, radiatum, pyramidal of CA3). Five days after lesion [ 3 H]-glutamate binding increased significantly in the CA3 region of the hippocampus but was not different from sham animals in the other hippocampal layers. Two weeks after lesion [ 3 H]-glutamate binding to quisqualate-insensitive receptors was increased in all the hippocampal layers, while kainate and quisqualate-sensitive receptors were not affected. These data are consistent with the possibility that 5-HT is a direct positive modulator of glutamate receptor subtypes

Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

DEFF Research Database (Denmark)

Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

2012-01-01

CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

Antibiotic modulation of the plasminogen binding ability of viridans group streptococci.

Science.gov (United States)

Teles, Cristina; Smith, Andrew; Lang, Sue

2012-01-01

The ability of viridans group streptococci to bind human plasminogen and its subsequent activation into plasmin may contribute to the pathogenesis of infective endocarditis (IE) by leading to a decreased stability of the streptococcal vegetation and facilitating dehiscence of emboli. At levels greater than or equal to their MICs, penicillin, vancomycin, and linezolid are efficacious in the treatment of streptococcal endocarditis. However, at sub-MICs, antibiotics can modulate the expression of bacterial genes, including virulence-associated genes, which can have counterproductive effects on the treatment of endocarditis. The effects of 1/8× and 1/4× MICs of penicillin, vancomycin, and linezolid on the plasminogen binding ability of IE isolates Streptococcus mitis 881/956, Streptococcus oralis 12601, and Streptococcus sanguinis 12403 were assessed phenotypically and the expression of plasminogen receptors α-enolase and glyceraldehyde 3-phosphate dehydrogenase of S. oralis 12601 when exposed to 1/4× MIC of penicillin, was analyzed through quantitative reverse transcription (qRT)-PCR. The plasminogen binding ability of S. mitis 881/956 and S. sanguinis 12403 remained unaffected by exposure to sub-MICs of all of the antibiotics tested, while that of S. oralis 12601 was significantly enhanced by all of the antibiotics tested at sub-MICs. qRT-PCR analysis of S. oralis 12601 demonstrated an upregulation of the eno and gapdh genes, indicating an overexpression of plasminogen receptors. These findings suggest that for some endocarditis isolates, the effect of antibiotic sub-MICs, in addition to a reduced antibacterial effect, may influence the clinical response to nonsurgical therapy. It remains difficult to accurately predict isolate responses to sub-MIC antimicrobials since there appears to be interspecies variation.

ANALYSIS OF STRUCTURAL ELEMENT OF FAMILY 6 CARBOHYDRATE BINDING MODULE (CTCBM6B OF ALPHA-L-ARABINOFURANOSIDASE FROM CLOSTRIDIUM THERMOCELLUM

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Shadab Ahmed

2013-06-01

Full Text Available The amino acid sequence of a family 6 carbohydrate binding module (CtCBM6B from Clostridium thermocellum alpha-L-arabinofuranosidase showed close evolutionary relationship with some other member of family 6 carbohydrate binding modules. The CD spectrum analysis confirmed the secondary structure prediction of CtCBM6B as both showed beta-sheets (44-48% and random coils (52-54% and no alpha-helix. The hydrogen bonding plot of CtCBM6B showed many segments of parallel and anti-parallel beta-strands which was similar to the secondary structure prediction by PSIPRED VIEW. The three dimensional structure of CtCBM6B generated by MODELLER revealed a typical beta-sandwich architecture at its core, characteristic of beta-jelly roll CBM superfamily. The Ramachandran plot analysis by PROCHECK showed that out of 134 residues, 92.9% were in most favoured region, 6.2% in additionally allowed region and only 0.9% in generously allowed region which indicated a stable conformation of 3D model of CtCBM6B. The docking analysis of CtCBM6B for finding putative ligand binding sites showed that it has high binding affinity for arabinobiose, beta-L-arabinofuranose and beta-D-xylopyranose indicated by lower ligand binding energy (-14.28 kcal mol–1, -12.5 kcal mol–1 and -11.3 kcal mol–1, respectively. CtCBM6B also showed appreciable binding affinity with alpha-D-xylopyranose (–10.8 kcal mol–1, beta-L-arabinopyranose (–10.2 kcal mol-1, alpha-L-arabinopyranose (–10.0 kcal mol–1 and alpha-L-arabinofuranose (–8.75 kcal mol–1. The results indicated that CtCBM6B has high potential for binding arabinan, xylans and substituted xylans.

Statistical significance of cis-regulatory modules

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Smith Andrew D

2007-01-01

Full Text Available Abstract Background It is becoming increasingly important for researchers to be able to scan through large genomic regions for transcription factor binding sites or clusters of binding sites forming cis-regulatory modules. Correspondingly, there has been a push to develop algorithms for the rapid detection and assessment of cis-regulatory modules. While various algorithms for this purpose have been introduced, most are not well suited for rapid, genome scale scanning. Results We introduce methods designed for the detection and statistical evaluation of cis-regulatory modules, modeled as either clusters of individual binding sites or as combinations of sites with constrained organization. In order to determine the statistical significance of module sites, we first need a method to determine the statistical significance of single transcription factor binding site matches. We introduce a straightforward method of estimating the statistical significance of single site matches using a database of known promoters to produce data structures that can be used to estimate p-values for binding site matches. We next introduce a technique to calculate the statistical significance of the arrangement of binding sites within a module using a max-gap model. If the module scanned for has defined organizational parameters, the probability of the module is corrected to account for organizational constraints. The statistical significance of single site matches and the architecture of sites within the module can be combined to provide an overall estimation of statistical significance of cis-regulatory module sites. Conclusion The methods introduced in this paper allow for the detection and statistical evaluation of single transcription factor binding sites and cis-regulatory modules. The features described are implemented in the Search Tool for Occurrences of Regulatory Motifs (STORM and MODSTORM software.

Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

DEFF Research Database (Denmark)

Damgaard, Maria; Al-Khawaja, Anas; Nittegaard-Nielsen, Mia

2017-01-01

-affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined......-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have...... δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high...

Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

Science.gov (United States)

Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

2011-09-08

Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation*

Science.gov (United States)

Almenar-Queralt, Angels; Kim, Sonia N.; Benner, Christopher; Herrera, Cheryl M.; Kang, David E.; Garcia-Bassets, Ivan; Goldstein, Lawrence S. B.

2013-01-01

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment. PMID:24145027

Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

Science.gov (United States)

Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

2013-12-06

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

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.

Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

Science.gov (United States)

Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

2014-01-01

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

FHA domains as phospho-threonine binding modules in cell signaling.

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Hammet, Andrew; Pike, Brietta L; McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2003-01-01

Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.

Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

Science.gov (United States)

Nieto, Lidia; Tharun, Inga M; Balk, Mark; Wienk, Hans; Boelens, Rolf; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

2015-11-20

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio.

Science.gov (United States)

Weidmann, Chase A; Qiu, Chen; Arvola, René M; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2016-08-02

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

Shielding voices: The modulation of binding processes between voice features and response features by task representations.

Science.gov (United States)

Bogon, Johanna; Eisenbarth, Hedwig; Landgraf, Steffen; Dreisbach, Gesine

2017-09-01

Vocal events offer not only semantic-linguistic content but also information about the identity and the emotional-motivational state of the speaker. Furthermore, most vocal events have implications for our actions and therefore include action-related features. But the relevance and irrelevance of vocal features varies from task to task. The present study investigates binding processes for perceptual and action-related features of spoken words and their modulation by the task representation of the listener. Participants reacted with two response keys to eight different words spoken by a male or a female voice (Experiment 1) or spoken by an angry or neutral male voice (Experiment 2). There were two instruction conditions: half of participants learned eight stimulus-response mappings by rote (SR), and half of participants applied a binary task rule (TR). In both experiments, SR instructed participants showed clear evidence for binding processes between voice and response features indicated by an interaction between the irrelevant voice feature and the response. By contrast, as indicated by a three-way interaction with instruction, no such binding was found in the TR instructed group. These results are suggestive of binding and shielding as two adaptive mechanisms that ensure successful communication and action in a dynamic social environment.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

Science.gov (United States)

Hsiao, An-Shan; Xue, Yan

2017-01-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells

International Nuclear Information System (INIS)

Gallo, V.; Wise, B.C.; Vaccarino, F.; Guidotti, A.

1985-01-01

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, the authors have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [ 35 S]TBPS. At 25 degrees C, and in a modified Locke solution, the [ 35 S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [ 35 S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [ 35 S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [ 35 S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [ 35 S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [ 35 S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam

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.

Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target.

Science.gov (United States)

Imbert, Isabelle; Botto, Jean-Marie; Farra, Claude D; Domloge, Nouha

2012-06-01

Telomere shortening is considered as one of the main characteristics of cellular aging by limiting cellular division. Besides the fundamental advances through the discoveries of telomere and telomerase, which were recognized by a Nobel Prize, telomere protection remains an essential area of research. Recently, it was evidenced that studying the cross-talks between the proteins associated with telomere should provide a better understanding of the mechanistic basis for telomere-associated aging phenotypes. In this review, we discuss the current knowledge on telomere shortening, telomerase activity, and the essential role of telomere binding proteins in telomere stabilization and telomere-end protection. This review highlights the capacity of telomere binding proteins to limit cellular senescence and to maintain skin tissue homeostasis, which is of key importance to reduce accelerated tissue aging. Future studies addressing telomere protection and limitation of DNA damage response in human skin should include investigations on telomere binding proteins. As little is known about the expression of telomere binding proteins in human skin and modulation of their expression with aging, it remains an interesting field of skin research and a key area for future skin protection and anti-aging developments. © 2012 Wiley Periodicals, Inc.

A novel fibronectin binding motif in MSCRAMMs targets F3 modules.

Directory of Open Access Journals (Sweden)

Sabitha Prabhakaran

Full Text Available BBK32 is a surface expressed lipoprotein and fibronectin (Fn-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21-205 of the lipoprotein.Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence.We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.

Simulation analysis of the cellulase Cel7A carbohydrate binding module on the surface of the cellulose Iβ

Energy Technology Data Exchange (ETDEWEB)

Alekozai, Emal M. [Univ. of Heidelberg (Germany); Univ. of Tennessee, Knoxville, TN (United States); GhattyVenkataKrishna, Pavan K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Uberbacher, Edward C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Crowley, Michael F. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States); Smith, Jeremy C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Cheng, Xiaolin [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)

2013-08-22

The Family 7 cellobiohydrolase (Cel7A) from Trichoderma reesei consists of a carbohydrate-binding module (CBM) joined by a linker to a catalytic domain. Cellulose hydrolysis is limited by the accessibility of Cel7A to crystalline substrates, which is perceived to be primarily mediated by the CBM. The binding of CBM to the cellulose I fiber is characterized by combined Brownian dynamics (BD) and molecular dynamics (MD) simulations. Our results confirm that CBM prefers to dock to the hydrophobic than to the hydrophilic fiber faces. Both electrostatic (ES) and van der Waals (VDW) interactions are required for achieving the observed binding preference. The VDW interactions play a more important role in stabilizing the CBM-fiber binding, whereas the ES interactions contribute through the formation of a number of hydrogen bonds between the CBM and the fiber. At long distances, an ES steering effect is also observed that tends to align the CBM in an antiparallel manner relative to the fiber axis. Moreover, the MD results reveal hindered diffusion of the CBM on all fiber surfaces. The binding of the CBM to the hydrophobic surfaces is found to involve partial dewetting at the CBM-fiber interface coupled with local structural arrangements of the protein. The present simulation results complement and rationalize a large body of previous work and provide detailed insights into the mechanism of the CBM-cellulose fiber interactions.

Advanced technologies for plant cell wall evolution and diversity

DEFF Research Database (Denmark)

Fangel, Jonatan Ulrik

Plant cell walls consist of polysaccharides, glycoproteins and phenolic polymers interlinked together in a highly complex network. The detailed analysis of cell walls is challenging because of their inherent complexity and heterogeneity. Also, complex carbohydrates, unlike proteins and nucleotides...... cannot really be synthesised or sequenced. The work described in this thesis is focused to a large extent on the development of a microarray-based high-throughput method for cell wall analysis known as Comprehensive microarray polymer profiling or CoMPP. The procedure uses highly specific molecular...... probes (monoclonal antibodies mAbs and carbohydrate binding modules, CBMs) to rapidly profile polysaccharides across a sample set. During my PhD I have further developed the CoMPP technique and used it for cell wall analysis within the context of a variety of applied and fundamental projects. The data...

Cell wall regeneration in Bangia atropurpurea (Rhodophyta) protoplasts observed using a mannan-specific carbohydrate-binding module.

Science.gov (United States)

Umemoto, Yoshiaki; Araki, Toshiyoshi

2010-02-01

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (beta-1,4-mannan, beta-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized beta-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of beta-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP-CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP-CBM27 maintained its binding ability to soluble beta-mannans, while normal GFP could not bind to beta-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP-CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete beta-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, beta-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP-CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP-CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of beta-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.

pH modulates the binding of early growth response protein 1 transcription factor to DNA.

Science.gov (United States)

Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

2013-08-01

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. © 2013 FEBS.

Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2

DEFF Research Database (Denmark)

Krintel, Christian; Francotte, Pierre; Pickering, Darryl S

2016-01-01

of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser...... of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from −4.9 (2) and −7.5 (3) to −6.2 (4) and −14.5 (5), but also a less favorable binding entropy (−TΔS, kcal/mol) from −2.3 (2) and −1.3 (3) to −0......-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation...

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

DEFF Research Database (Denmark)

Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

2017-01-01

is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super......-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate...

Characterisation of the effect of ion channel modulators on I1-imidazoline binding sites in bovine adrenal medulla

International Nuclear Information System (INIS)

Musgrave, I.F.; Kotsopoulos, D.; Hughes, R.A.

1998-01-01

Full text: The structure of I 1 -imidazoline binding sites is still unknown and we have proposed that they represent ion channels (i). In these experiments we characterised the effects of the known ion channel modulators methyltriphenylphosphonium (MTPP), 4-aminopyridine (4-AP) and tetraethyl ammonium (TEA) on [ 3 H] clonidine binding in bovine adrenal medullary membranes as these membranes have a relatively well defined I 1 -imidazoline binding site (Molderings et al, 1993). Membranes from bovine adrenal medulla's were prepared by a minor modification of the method of Rapier et al. [ 3 H] Clonidine binding was performed by the method of Ernsberger et al (3), with [ 3 H] clonidine (62 Ci/mmol) used at a final concentration of 5 nM. [ 3 H] Clonidine binding was displaced from bovine adrenal medullary membranes by adrenergic drugs with the order of potency being oxymetazoline > clonidine > moxonidine = idazoxan >> yonimbine. This order of potency is consistent with previous studies of I 1 -imidazoline binding sites (4). Non-linear curve fitting to this data was consistent with a single site model. Both TEA and 4-AP displaced [ H] clonidine with similar potency to its effect on ion channels, TEA having a EC>> of 54 ± 0.3 μM (n=3). The displacement of [ 3 H] clonidine produced by both TEA and 4-AP also fitted to a single site model. Displacement of [ 3 H] clonidine by MTPP fitted a two site model (p 1 -imidazoline binding sites defined with [ 3 H] clonidine may represent ion channels. We have used this data to perform molecular modelling and have determined a common conformation of I 1 -prefering ligands which will aid in the development of I 1 -selective ligands in the future. Copyright (1998) Australian Neuroscience Society

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Science.gov (United States)

Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

2012-01-01

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

Energy Technology Data Exchange (ETDEWEB)

Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: dzajonc@liai.org [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)

2014-03-01

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host�

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

International Nuclear Information System (INIS)

Nemčovičová, Ivana; Zajonc, Dirk M.

2014-01-01

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X 6 G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT genomic binding patterns discerns cell-specific cis-regulatory modules

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Kang Keunsoo

2013-01-01

Full Text Available Abstract Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM, which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

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Liu, Peng; Stajich, Jason E

2015-04-01

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of secondary binding sites in proteins using fragment screening.

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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.

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

Energy Technology Data Exchange (ETDEWEB)

Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T.; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.

2016-08-02

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

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Abramyan, John; Stajich, Jason E

2012-01-01

Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

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Kishore Polireddy

Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Bitopic Ligands and Metastable Binding Sites

DEFF Research Database (Denmark)

Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

2017-01-01

of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

International Nuclear Information System (INIS)

Rodriguez-Munoz, Rafael; Villarreal-Silva, Marcela; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Mondragon, Monica; Mondragon, Ricardo; Cerna, Joel; Cisneros, Bulmaro

2008-01-01

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation

The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

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Jörg Lucas

Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

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H. S. Euzger

1999-01-01

Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

What Happened to the IGF Binding Proteins?

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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.

Cellular bone matrices: viable stem cell-containing bone graft substitutes.

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Skovrlj, Branko; Guzman, Javier Z; Al Maaieh, Motasem; Cho, Samuel K; Iatridis, James C; Qureshi, Sheeraz A

2014-11-01

Advances in the field of stem cell technology have stimulated the development and increased use of allogenic bone grafts containing live mesenchymal stem cells (MSCs), also known as cellular bone matrices (CBMs). It is estimated that CBMs comprise greater than 17% of all bone grafts and bone graft substitutes used. To critically evaluate CBMs, specifically their technical specifications, existing published data supporting their use, US Food and Drug Administration (FDA) regulation, cost, potential pitfalls, and other aspects pertaining to their use. Areview of literature. A series of Ovid, Medline, and Pubmed-National Library of Medicine/National Institutes of Health (www.ncbi.nlm.nih.gov) searches were performed. Only articles in English journals or published with English language translations were included. Level of evidence of the selected articles was assessed. Specific technical information on each CBM was obtained by direct communication from the companies marketing the individual products. Five different CBMs are currently available for use in spinal fusion surgery. There is a wide variation between the products with regard to the average donor age at harvest, total cellular concentration, percentage of MSCs, shelf life, and cell viability after defrosting. Three retrospective studies evaluating CBMs and fusion have shown fusion rates ranging from 90.2% to 92.3%, and multiple industry-sponsored trials are underway. No independent studies evaluating spinal fusion rates with the use of CBMs exist. All the commercially available CBMs claim to meet the FDA criteria under Section 361, 21 CFR Part 1271, and are not undergoing FDA premarket review. The CBMs claim to provide viable MSCs and are offered at a premium cost. Numerous challenges exist in regard to MSCs' survival, function, osteoblastic potential, and cytokine production once implanted into the intended host. Cellular bone matrices may be a promising bone augmentation technology in spinal fusion surgery

Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation▿

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Sanders, Steven L.; Arida, Ahmad R.; Phan, Funita P.

2010-01-01

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability. PMID:20679488

Requirement for the phospho-H2AX binding module of Crb2 in double-strand break targeting and checkpoint activation.

Science.gov (United States)

Sanders, Steven L; Arida, Ahmad R; Phan, Funita P

2010-10-01

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.

A web server for analysis, comparison and prediction of protein ligand binding sites.

Science.gov (United States)

Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

2016-03-25

One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

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Garima Tiwari

Full Text Available Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i or K(d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i or K(d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors

Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).

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Zhou, Li; Yeo, Alan T; Ballarano, Carmine; Weber, Urs; Allen, Karen N; Gilmore, Thomas D; Whitty, Adrian

2014-12-23

Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small-molecule inhibitors.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

Science.gov (United States)

Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

2017-01-01

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

Allosteric modulation of G-protein coupled receptors

DEFF Research Database (Denmark)

Jensen, Anders A.; Spalding, Tracy A

2004-01-01

are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

The TROVE module: a common element in Telomerase, Ro and Vault ribonucleoproteins.

Science.gov (United States)

Bateman, Alex; Kickhoefer, Valerie

2003-10-16

Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. The TROVE module--Telomerase, Ro and Vault module--is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins.

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

Science.gov (United States)

Brauser, Annemarie; Schroeder, Indra; Gutsmann, Thomas; Cosentino, Cristian; Moroni, Anna; Winterhalter, Mathias

2012-01-01

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg2+. Analysis of the resulting amplitude histograms with β distributions revealed the rate constants of blocking (kOB) and unblocking (kBO) in the range of 1,000 to 120,000 s−1. As expected for a bimolecular reaction, kOB was proportional to blocker concentration and kBO independent of it. kOB was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being ∼25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg2+ and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg2+. The difference in the accessibility of the binding sites also explains the dependency of kOB on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding. PMID:22689827

Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

Science.gov (United States)

Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

2013-07-01

Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

Crystal structure of CbpF, a bifunctional choline-binding protein and autolysis regulator from Streptococcus pneumoniae.

Science.gov (United States)

Molina, Rafael; González, Ana; Stelter, Meike; Pérez-Dorado, Inmaculada; Kahn, Richard; Morales, María; Moscoso, Miriam; Campuzano, Susana; Campillo, Nuria E; Mobashery, Shahriar; García, José L; García, Pedro; Hermoso, Juan A

2009-03-01

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline-binding proteins. The three-dimensional structure of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non-consensus choline-binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well-defined modules. The carboxy-terminal module is involved in cell wall binding and the amino-terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.

Effects of flow changes on radiotracer binding: Simultaneous measurement of neuroreceptor binding and cerebral blood flow modulation.

Science.gov (United States)

Sander, Christin Y; Mandeville, Joseph B; Wey, Hsiao-Ying; Catana, Ciprian; Hooker, Jacob M; Rosen, Bruce R

2017-01-01

The potential effects of changes in blood flow on the delivery and washout of radiotracers has been an ongoing question in PET bolus injection studies. This study provides practical insight into this topic by experimentally measuring cerebral blood flow (CBF) and neuroreceptor binding using simultaneous PET/MRI. Hypercapnic challenges (7% CO 2 ) were administered to non-human primates in order to induce controlled increases in CBF, measured with pseudo-continuous arterial spin labeling. Simultaneously, dopamine D 2 /D 3 receptor binding of [ 11 C]raclopride or [ 18 F]fallypride was monitored with dynamic PET. Experiments showed that neither time activity curves nor quantification of binding through binding potentials ( BP ND ) were measurably affected by CBF increases, which were larger than two-fold. Simulations of experimental procedures showed that even large changes in CBF should have little effect on the time activity curves of radiotracers, given a set of realistic assumptions. The proposed method can be applied to experimentally assess the flow sensitivity of other radiotracers. Results demonstrate that CBF changes, which often occur due to behavioral tasks or pharmacological challenges, do not affect PET [ 11 C]raclopride or [ 18 F]fallypride binding studies and their quantification. The results from this study suggest flow effects may have limited impact on many PET neuroreceptor tracers with similar properties.

The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist.

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Garret Suen

Full Text Available Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs, carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

International Nuclear Information System (INIS)

Murphy, R.B.; Schuster, D.I.

1982-01-01

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [ 3 H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [ 3 H]-dopamine binding indicates an IC50 for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [ 3 H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems

Cerebellar malformations alter regional cerebral development.

Science.gov (United States)

Bolduc, Marie-Eve; Du Plessis, Adre J; Evans, Alan; Guizard, Nicolas; Zhang, Xun; Robertson, Richard L; Limperopoulos, Catherine

2011-12-01

The aim of this study was to compare total and regional cerebral volumes in children with isolated cerebellar malformations (CBMs) with those in typically developing children, and to examine the extent to which cerebellar volumetric reductions are associated with total and regional cerebral volumes. This is a case-control study of children diagnosed with isolated CBMs. Each child was matched on age and sex to two typically developing children. Using advanced three-dimensional volumetric magnetic resonance imaging, the cerebrum was segmented into tissue classes and partitioned into eight regions. Analysis of variance was used to compare cerebral volumes between children with CBMs and control children, and linear regressions to examine the impact of cerebellar volume reduction on cerebral volumes. Magnetic resonance imaging was performed at a mean age of 27 months in 20 children (10 males, 10 females) with CBMs and 40 typically developing children. Children with CBMs showed significantly smaller deep grey matter nuclei (p developing children. Greater cerebellar volumetric reduction in children with CBMs was associated with decreased total cerebral volume and deep grey matter nuclei (p = 0.02), subgenual white/grey matter (p = 0.001), midtemporal white (p = 0.02) and grey matter (p = 0.01), and parieto-occipital grey matter (p = 0.004). CBMs are associated with impaired regional cerebral growth, suggesting deactivation of principal cerebello-cerebral pathways. © The Authors. Developmental Medicine & Child Neurology © 2011 Mac Keith Press.

A general model of confidence building: analysis and implications

International Nuclear Information System (INIS)

Kilgour, D.M.

1998-01-01

For more than two decades, security approaches in Europe have included confidence building. Many have argued that Confidence-Building Measures (CBMS) played an essential role in the enormous transformations that took place there. Thus, it is hardly,surprising that CBMs have been proposed as measures to reduce tensions and transform security relationships elsewhere in the world. The move toward wider application of CBMs has strengthened recently, as conventional military, diplomatic, and humanitarian approaches seem to have failed to address problems associated with peace-building and peace support operations. There is, however, a serious problem. We don't really know why, or even how, CBMs work. Consequently, we have no reliable way to design CBMs that would be appropriate in substance, form, and timing for regions culturally, geographically, and militarily different from Europe. Lacking a solid understanding of confidence building, we are handicapped in our efforts to extend its successes to the domain of peace building and peace support. To paraphrase Macintosh, if we don't know how CBMs succeeded in the past, then we are unlikely to be good at maintaining, improving, or extending them. The specific aim of this project is to step into this gap, using the methods of game theory to clarify some aspects of the underlying logic of confidence building. Formal decision models will be shown to contribute new and valuable insights that will assist in the design of CBMs to contribute to new problems and in new arenas. (author)

1H, 15N and 13C backbone and side-chain resonance assignments of a family 32 carbohydrate-binding module from the Clostridium perfringens NagH.

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Grondin, Julie M; Chitayat, Seth; Ficko-Blean, Elizabeth; Boraston, Alisdair B; Smith, Steven P

2012-10-01

The Gram-positive anaerobe Clostridium perfringens is an opportunistic bacterial pathogen that secretes a battery of enzymes involved in glycan degradation. These glycoside hydrolases are thought to be involved in turnover of mucosal layer glycans, and in the spread of major toxins commonly associated with the development of gastrointestinal diseases and gas gangrene in humans. These enzymes employ multi-modularity and carbohydrate-binding function to degrade extracellular eukaryotic host sugars. Here, we report the full (1)H, (15)N and (13)C chemical shift resonance assignments of the first family 32 carbohydrate-binding module from NagH, a secreted family 84 glycoside hydrolase.

Bindings in working memory: The role of object-based attention.

Science.gov (United States)

Gao, Zaifeng; Wu, Fan; Qiu, Fangfang; He, Kaifeng; Yang, Yue; Shen, Mowei

2017-02-01

Over the past decade, it has been debated whether retaining bindings in working memory (WM) requires more attention than retaining constituent features, focusing on domain-general attention and space-based attention. Recently, we proposed that retaining bindings in WM needs more object-based attention than retaining constituent features (Shen, Huang, & Gao, 2015, Journal of Experimental Psychology: Human Perception and Performance, doi: 10.1037/xhp0000018 ). However, only unitized visual bindings were examined; to establish the role of object-based attention in retaining bindings in WM, more emperical evidence is required. We tested 4 new bindings that had been suggested requiring no more attention than the constituent features in the WM maintenance phase: The two constituent features of binding were stored in different WM modules (cross-module binding, Experiment 1), from auditory and visual modalities (cross-modal binding, Experiment 2), or temporally (cross-time binding, Experiments 3) or spatially (cross-space binding, Experiments 4-6) separated. In the critical condition, we added a secondary object feature-report task during the delay interval of the change-detection task, such that the secondary task competed for object-based attention with the to-be-memorized stimuli. If more object-based attention is required for retaining bindings than for retaining constituent features, the secondary task should impair the binding performance to a larger degree relative to the performance of constituent features. Indeed, Experiments 1-6 consistently revealed a significantly larger impairment for bindings than for the constituent features, suggesting that object-based attention plays a pivotal role in retaining bindings in WM.

The TROVE module: A common element in Telomerase, Ro and Vault ribonucleoproteins

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Bateman Alex

2003-10-01

Full Text Available Abstract Background Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. Results The TROVE module – Telomerase, Ro and Vault module – is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. Conclusions This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins.

Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

KAUST Repository

Li, Lixin

2013-07-01

Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

Science.gov (United States)

Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

2016-05-09

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity

HuR/ELAVL1 RNA binding protein modulates interleukin-8 induction by muco-active ribotoxin deoxynivalenol

International Nuclear Information System (INIS)

Choi, Hye Jin; Yang, Hyun; Park, Seong Hwan; Moon, Yuseok

2009-01-01

HuR/Elav-like RNA binding protein 1 (ELAVL1) positively regulates mRNA stability of AU-rich elements (ARE)-containing transcript such as pro-inflammatory cytokines. Ribotoxic stresses can trigger the production of pro-inflammatory mediators by enhancing mRNA stability and the transcriptional activity. We investigated the effects of ribotoxin deoxynivalenol (DON) on HuR translocation and its involvement in the regulation of the pro-inflammatory interleukin-8 (IL-8) mRNA stability. Exposure to the muco-active DON induced nuclear export of both endogenous and exogenous HuR RNA binding protein in human intestinal epithelial cells. Moreover, the interference with HuR protein production suppressed ribotoxic DON-induced IL-8 secretion and its mRNA stability. Cytoplasmic HuR protein interacted with IL-8 mRNA and the complex stabilization was due to the presence of 3'-untranslated region of the transcript. Partly in terms of IL-8-modulating transcription factors, HuR protein was demonstrated to be positively and negatively associated with DON-induced early growth response gene 1 (EGR-1) and activating transcription factor 3 (ATF3), respectively. HuR was a critical mechanistic link between ribotoxic stress and the pro-inflammatory cytokine production, and may have a broader functional significance with regard to mucosal insults since ribotoxic stress responses are also produced upon interactions with the diverse environment of gut.

Does positive selection drive transcription factor binding site turnover? A test with Drosophila cis-regulatory modules.

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Bin Z He

2011-04-01

Full Text Available Transcription factor binding site(s (TFBS gain and loss (i.e., turnover is a well-documented feature of cis-regulatory module (CRM evolution, yet little attention has been paid to the evolutionary force(s driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.

Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

International Nuclear Information System (INIS)

Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni; Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin; Jiang, Tao

2006-01-01

Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents

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.

The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

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Clara L Oeste

2011-01-01

Full Text Available Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184 in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14-PGJ(2 (15d-PGJ(2 and Δ(12-PGJ(2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO, a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.

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Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

2012-08-24

Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.

Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

Energy Technology Data Exchange (ETDEWEB)

Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

2007-01-01

The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

Science.gov (United States)

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Modulation of SHBG binding to testosterone and estradiol by sex and morbid obesity.

Science.gov (United States)

Grasa, María Del Mar; Gulfo, José; Camps, Núria; Alcalá, Rosa; Monserrat, Laura; Moreno-Navarrete, José María; Ortega, Francisco José; Esteve, Montserrat; Remesar, Xavier; Fernández-López, José Antonio; Fernández-Real, José Manuel; Alemany, Marià

2017-04-01

Sex hormone-binding globulin (SHBG) binds and transports testosterone and estradiol in plasma. The possibility that SHBG is a mixture of transporting proteins has been postulated. We analyzed in parallel the effects of obesity status on the levels and binding capacity of circulating SHBG and their relationship with testosterone and estradiol. Anthropometric measures and plasma were obtained from apparently healthy young (i.e. 35 ± 7 years) premenopausal women ( n =  32) and men ( n =  30), with normal weight and obesity (BMI >30 kg/m 2 ). SHBG protein (Western blot), as well as the plasma levels of testosterone, estradiol, cortisol and insulin (ELISA) were measured. Specific binding of estradiol and testosterone to plasma SHBG was analyzed using tritium-labeled hormones. Significant differences in SHBG were observed within the obesity status and gender, with discordant patterns of change in testosterone and estradiol. In men, testosterone occupied most of the binding sites. Estrogen binding was much lower in all subjects. Lower SHBG of morbidly obese (BMI >40 kg/m 2 ) subjects affected testosterone but not estradiol. The ratio of binding sites to SHBG protein levels was constant for testosterone, but not for estradiol. The influence of gender was maximal in morbid obesity, with men showing the highest binding / SHBG ratios. The results reported here are compatible with SHBG being a mixture of at least two functionally different hormone-binding globulins, being affected by obesity and gender and showing different structure, affinities for testosterone and estradiol and also different immunoreactivity. © 2017 European Society of Endocrinology.

Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo.

Science.gov (United States)

Hwang, Geelsu; Liu, Yuan; Kim, Dongyeop; Li, Yong; Krysan, Damian J; Koo, Hyun

2017-06-01

Candida albicans is frequently detected with heavy infection by Streptococcus mutans in plaque-biofilms from children with early-childhood caries (ECC). This cross-kingdom biofilm contains an extensive matrix of extracellular α-glucans that is produced by an exoenzyme (GtfB) secreted by S. mutans. Here, we report that mannans located on the outer surface of C. albicans cell-wall mediates GtfB binding, enhancing glucan-matrix production and modulating bacterial-fungal association within biofilms formed in vivo. Using single-molecule atomic force microscopy, we determined that GtfB binds with remarkable affinity to mannans and to the C. albicans surface, forming a highly stable and strong bond (1-2 nN). However, GtfB binding properties to C. albicans was compromised in strains defective in O-mannan (pmt4ΔΔ) or N-mannan outer chain (och1ΔΔ). In particular, the binding strength of GtfB on och1ΔΔ strain was severely disrupted (>3-fold reduction vs. parental strain). In turn, the GtfB amount on the fungal surface was significantly reduced, and the ability of C. albicans mutant strains to develop mixed-species biofilms with S. mutans was impaired. This phenotype was independent of hyphae or established fungal-biofilm regulators (EFG1, BCR1). Notably, the mechanical stability of the defective biofilms was weakened, resulting in near complete biomass removal by shear forces. In addition, these in vitro findings were confirmed in vivo using a rodent biofilm model. Specifically, we observed that C. albicans och1ΔΔ was unable to form cross-kingdom biofilms on the tooth surface of rats co-infected with S. mutans. Likewise, co-infection with S. mutans defective in GtfB was also incapable of forming mixed-species biofilms. Taken together, the data support a mechanism whereby S. mutans-secreted GtfB binds to the mannan layer of C. albicans to promote extracellular matrix formation and their co-existence within biofilms. Enhanced understanding of GtfB-Candida interactions

The theory of confidence-building measures

International Nuclear Information System (INIS)

Darilek, R.E.

1992-01-01

This paper discusses the theory of Confidence-Building Measures (CBMs) in two ways. First, it employs a top-down, deductively oriented approach to explain CBM theory in terms of the arms control goals and objectives to be achieved, the types of measures to be employed, and the problems or limitations likely to be encountered when applying CBMs to conventional or nuclear forces. The chapter as a whole asks how various types of CBMs might function during a political - military escalation from peacetime to a crisis and beyond (i.e. including conflict), as well as how they might operate in a de-escalatory environment. In pursuit of these overarching issues, the second section of the chapter raises a fundamental but complicating question: how might the next all-out war actually come aoubt - by unpremeditated escalation resulting from misunderstanding or miscalculation, or by premeditation resulting in a surprise attack? The second section of the paper addresses this question, explores its various implications for CBMs, and suggests the potential contribution of different types of CBMs toward successful resolution of the issues involved

Fighting Poverty with Facts: Community-Based Monitoring Systems

International Development Research Centre (IDRC) Digital Library (Canada)

2009-01-01

Jan 1, 2009 ... Documents and Articles: ... This book presents the Community-Based Monitoring System ( CBMS ) ... Drawing from CBMS experience in Africa and Asia, the authors present recommendations for policymakers, donor agencies ...

Multivalent Soluble Antigen Arrays Exhibit High Avidity Binding and Modulation of B Cell Receptor-Mediated Signaling to Drive Efficacy against Experimental Autoimmune Encephalomyelitis.

Science.gov (United States)

Hartwell, Brittany L; Pickens, Chad J; Leon, Martin; Berkland, Cory

2017-06-12

A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgA PLP:LABL ), consisting of a hyaluronic acid (HA) linear polymer backbone cografted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous studies established that hydrolyzable SAgA PLP:LABL , employing a degradable linker to codeliver PLP and LABL, was therapeutic in experimental autoimmune encephalomyelitis (EAE) in vivo and exhibited antigen-specific binding with B cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling in vitro. Our results pointed to sustained BCR engagement as the SAgA PLP:LABL therapeutic mechanism, so we developed a new version of the SAgA molecule using nonhydrolyzable conjugation chemistry, hypothesizing it would enhance and maintain the molecule's action at the cell surface to improve efficacy. "Click SAgA" (cSAgA PLP:LABL ) uses hydrolytically stable covalent conjugation chemistry (Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC)) rather than a hydrolyzable oxime bond to attach PLP and LABL to HA. We explored cSAgA PLP:LABL B cell engagement and modulation of BCR-mediated signaling in vitro through flow cytometry binding and calcium flux signaling assays. Indeed, cSAgA PLP:LABL exhibited higher avidity B cell binding and greater dampening of BCR-mediated signaling than hydrolyzable SAgA PLP:LABL . Furthermore, cSAgA PLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgA PLP:LABL , achieving equivalent efficacy at one-quarter of the dose. These results indicate that nonhydrolyzable conjugation increased the avidity of cSAgA PLP:LABL to drive in vivo efficacy through modulated BCR-mediated signaling.

Impact of orientation of carbohydrate binding modules family 22 and 6 on the catalytic activity of Thermotoga maritima xylanase XynB.

Science.gov (United States)

Tajwar, Razia; Shahid, Saher; Zafar, Rehan; Akhtar, Muhammad Waheed

2017-11-01

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before. Copyright © 2017 Elsevier Inc. All rights reserved.

Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation.

Directory of Open Access Journals (Sweden)

Deanne W Sammond

Full Text Available Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs to carbohydrate-binding modules (CBMs. Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests

Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors - A Structural Perspective of Ligands and Mutants

DEFF Research Database (Denmark)

Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G

2015-01-01

modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different m......Glu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs....

Mua (HP0868) Is a Nickel-Binding Protein That Modulates Urease Activity in Helicobacter pylori

Science.gov (United States)

Benoit, Stéphane L.; Maier, Robert J.

2011-01-01

A novel mechanism aimed at controlling urease expression in Helicobacter pylori in the presence of ample nickel is described. Higher urease activities were observed in an hp0868 mutant (than in the wild type) in cells supplemented with nickel, suggesting that the HP0868 protein (herein named Mua for modulator of urease activity) represses urease activity when nickel concentrations are ample. The increase in urease activity in the Δmua mutant was linked to an increase in urease transcription and synthesis, as shown by quantitative real-time PCR, SDS-PAGE, and immunoblotting against UreAB. Increased urease synthesis was also detected in a Δmua ΔnikR double mutant strain. The Δmua mutant was more sensitive to nickel toxicity but more resistant to acid challenge than was the wild-type strain. Pure Mua protein binds 2 moles of Ni2+ per mole of dimer. Electrophoretic mobility shift assays did not reveal any binding of Mua to the ureA promoter or other selected promoters (nikR, arsRS, 5′ ureB-sRNAp). Previous yeast two-hybrid studies indicated that Mua and RpoD may interact; however, only a weak interaction was detected via cross-linking with pure components and this could not be verified by another approach. There was no significant difference in the intracellular nickel level between wild-type and mua mutant cells. Taken together, our results suggest the HP0868 gene product represses urease transcription when nickel levels are high through an as-yet-uncharacterized mechanism, thus counterbalancing the well-described NikR-mediated activation. PMID:21505055

Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

Science.gov (United States)

El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

2017-02-01

Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

H-1 and N-15 resonance assignment of the second fibronectin type III module of the neural cell adhesion molecule

DEFF Research Database (Denmark)

Kiselyov, Vladislav V; Berezin, Vladimir; Bock, Elisabeth

2008-01-01

We report here the NMR assignment of the second fibronectin type III module of the neural cell adhesion molecule (NCAM). This module has previously been shown to interact with the fibroblast growth factor receptor (FGFR), and the FGFR-binding site was mapped by NMR to the FG-loop region of the mo......We report here the NMR assignment of the second fibronectin type III module of the neural cell adhesion molecule (NCAM). This module has previously been shown to interact with the fibroblast growth factor receptor (FGFR), and the FGFR-binding site was mapped by NMR to the FG-loop region...... of the module. The FG-loop region also contains a putative nucleotide-binding motif, which was shown by NMR to interact with ATP. Furthermore, ATP was demonstrated to inhibit binding of the second F3 module of NCAM to FGFR....

Tight-binding electrons on triangular and kagome lattices under staggered modulated magnetic fields: quantum Hall effects and Hofstadter butterflies

International Nuclear Information System (INIS)

Li Juan; Wang Yifei; Gong Changde

2011-01-01

We consider the tight-binding models of electrons on a two-dimensional triangular lattice and kagome lattice under staggered modulated magnetic fields. Such fields have two components: a uniform-flux part with strength φ, and a staggered-flux part with strength Δφ. Various properties of the Hall conductances and Hofstadter butterflies are studied. When φ is fixed, variation of Δφ leads to the quantum Hall transitions and Chern numbers of Landau subbands being redistributed between neighboring pairs. The energy spectra with nonzero Δφs have similar fractal structures but quite different energy gaps compared with the original Hofstadter butterflies of Δφ = 0. Moreover, the fan-like structure of Landau levels in the low magnetic field region is also modified appreciably by Δφ.

Tight-binding electrons on triangular and kagome lattices under staggered modulated magnetic fields: quantum Hall effects and Hofstadter butterflies

Energy Technology Data Exchange (ETDEWEB)

Li Juan; Wang Yifei; Gong Changde, E-mail: yfwang_nju@hotmail.com [Center for Statistical and Theoretical Condensed Matter Physics, and Department of Physics, Zhejiang Normal University, Jinhua 321004 (China)

2011-04-20

We consider the tight-binding models of electrons on a two-dimensional triangular lattice and kagome lattice under staggered modulated magnetic fields. Such fields have two components: a uniform-flux part with strength {phi}, and a staggered-flux part with strength {Delta}{phi}. Various properties of the Hall conductances and Hofstadter butterflies are studied. When {phi} is fixed, variation of {Delta}{phi} leads to the quantum Hall transitions and Chern numbers of Landau subbands being redistributed between neighboring pairs. The energy spectra with nonzero {Delta}{phi}s have similar fractal structures but quite different energy gaps compared with the original Hofstadter butterflies of {Delta}{phi} = 0. Moreover, the fan-like structure of Landau levels in the low magnetic field region is also modified appreciably by {Delta}{phi}.

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

Science.gov (United States)

Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

2014-09-02

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes

KAUST Repository

Zhang, Meiling

2014-08-18

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT-04215 and BACOVA-04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xy-lose- configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM fromits homolog in the Prevotella bryantii B 14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. Aminimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

DEFF Research Database (Denmark)

Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas

2016-01-01

for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an i...... downstream of the 5' splice site can be blocked by SSOs to activate the exon. CONCLUSIONS: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease...

GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding.

Science.gov (United States)

Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic, Marko D; Guillet, Fabrice; Bouaita, Belkacem; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, Mouhssin

2018-01-01

In a screening of natural products for allosteric modulators of GABA A receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABA A and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

DEFF Research Database (Denmark)

Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav

2016-01-01

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn(2+) (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site......Terp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248(VI:13/6.48) microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism...

Molecular tweezers modulate 14-3-3 protein-protein interactions

Science.gov (United States)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Corticosterone binding to tissues of adrenalectomized lean and obese Zucker rats.

Science.gov (United States)

Grasa, M M; Cabot, C; Balada, F; Virgili, J; Sanchis, D; Monserrat, C; Fernández-López, J A; Remesar, X; Alemany, M

1998-12-01

The binding of corticosterone, dexamethasone and aldosterone was investigated in plasma and in homogenates of liver, kidney, brain, brown adipose tissue and visceral (periovaric) and subcutaneous white adipose tissues of Zucker lean and obese rats: intact controls, adrenalectomized and sham-operated. Corticosterone-binding globulin (CBG) accounted for most of the binding, whereas that of glucocorticoid and mineralocorticoid receptors was much lower. Plasma corticosterone levels increased in sham-operated and obviously decreased in the adrenalectomized animals. Sham-operated and adrenalectomized lean rats showed decreased plasma CBG; in the obese, CBG levels were lower than in controls and were not affected by either surgery. No variation with obesity or surgery was observed either in dexamethasone or aldosterone binding, the latter being practically zero in most samples. When expressed per unit of tissue protein, CBG activity was maximal in adipose tissues, with lowest values in brain and liver. In lean rats, tissue CBG activity decreased with either surgical treatment; no changes were observed in the obese, which also had lower CBG tissue levels. The relative lack of changes in CBG of obese rats suggests that they have lost -- at least in part -- the ability to counter-modulate the changes in glucocorticoid levels through CBG modulation, thus relying only on the control of corticosterone levels. This interpretation agrees with the postulated role of CBG modulating the availability of glucocorticoids to target cells.

Odorant binding protein 69a connects social interaction to modulation of social responsiveness in Drosophila.

Science.gov (United States)

Bentzur, Assa; Shmueli, Anat; Omesi, Liora; Ryvkin, Julia; Knapp, Jon-Michael; Parnas, Moshe; Davis, Fred P; Shohat-Ophir, Galit

2018-04-01

Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In Drosophila melanogaster, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. Although it is the most studied pheromone in flies, the mechanisms that determine the complexity of the response, its intensity and final output with respect to social context, sex and prior interaction, are still not well understood. Here we explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. Odorant binding protein 69a (Obp69a) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. We show that activation of cVA-sensing neurons is sufficeint to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies.

Special Attachments. Module 19.

Science.gov (United States)

South Carolina State Dept. of Education, Columbia. Office of Vocational Education.

This module on special attachments, one in a series dealing with industrial sewing machines, their attachments, and operation, covers four topics: gauges; cording attachment; zipper foot; and hemming, shirring, and binding. For each topic these components are provided: an introduction, directions, an objective, learning activities, student…

Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

Science.gov (United States)

Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

1983-10-01

Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

Phosphorylation Modulates Ameloblastin Self-assembly and Ca2+ Binding

Czech Academy of Sciences Publication Activity Database

Stakkestad, O.; Lyngstadaas, S. P.; Thiede, B.; Vondrášek, Jiří; Skalhegg, B. S.; Reseland, J. E.

2017-01-01

Roč. 8, Jul 27 (2017), č. článku 531. ISSN 1664-042X Institutional support: RVO:61388963 Keywords : ameloblastin * phosphorylation * self-assembly * Ca2+-binding * enamel * intrinsically disordered proteins Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.134, year: 2016 http://journal.frontiersin.org/article/10.3389/fphys.2017.00531/full

Locating Temporal Functional Dynamics of Visual Short-Term Memory Binding using Graph Modular Dirichlet Energy

Science.gov (United States)

Smith, Keith; Ricaud, Benjamin; Shahid, Nauman; Rhodes, Stephen; Starr, John M.; Ibáñez, Augustin; Parra, Mario A.; Escudero, Javier; Vandergheynst, Pierre

2017-02-01

Visual short-term memory binding tasks are a promising early marker for Alzheimer’s disease (AD). To uncover functional deficits of AD in these tasks it is meaningful to first study unimpaired brain function. Electroencephalogram recordings were obtained from encoding and maintenance periods of tasks performed by healthy young volunteers. We probe the task’s transient physiological underpinnings by contrasting shape only (Shape) and shape-colour binding (Bind) conditions, displayed in the left and right sides of the screen, separately. Particularly, we introduce and implement a novel technique named Modular Dirichlet Energy (MDE) which allows robust and flexible analysis of the functional network with unprecedented temporal precision. We find that connectivity in the Bind condition is less integrated with the global network than in the Shape condition in occipital and frontal modules during the encoding period of the right screen condition. Using MDE we are able to discern driving effects in the occipital module between 100-140 ms, coinciding with the P100 visually evoked potential, followed by a driving effect in the frontal module between 140-180 ms, suggesting that the differences found constitute an information processing difference between these modules. This provides temporally precise information over a heterogeneous population in promising tasks for the detection of AD.

Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

DEFF Research Database (Denmark)

Krintel, Christian; Frydenvang, Karla; Olsen, Lars

2012-01-01

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the ligand-binding domain and stabilize the agonist-bound conformation slow...

Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting.

Directory of Open Access Journals (Sweden)

Ann-Marie Chacko

Full Text Available Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1 facilitate targeted drug delivery to endothelial cells by "vascular immunotargeting." To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The "collaborative enhancement" of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The "collaborative enhancement" phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents.

Acetylcholinesterase potentiates [3H]fluorowillardiine and [3H]AMPA binding to rat cortical membranes

International Nuclear Information System (INIS)

Olivera, S.; Rodriguez-Ithurralde, D.; Henley, J.M.

1999-01-01

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[ 3 H]5-fluorowillardiine ([ 3 H]FW) and [ 3 H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to ∼60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [ 3 H]kainate binding. [ 3 H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC 50 values of 4x10 -5 and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [ 3 H]FW binding is Ca 2+ - and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [ 3 H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the B max of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

International Nuclear Information System (INIS)

Burrier, R.E.; Manson, C.R.; Brecher, P.

1987-01-01

The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. 14 C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell

Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

Energy Technology Data Exchange (ETDEWEB)

Burrier, R.E.; Manson, C.R.; Brecher, P.

1987-05-01

The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

Transcriptional regulation by competing transcription factor modules.

Directory of Open Access Journals (Sweden)

Rutger Hermsen

2006-12-01

Full Text Available Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input-output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits.

Bandstructure modulation for Si-h and Si-g nanotubes in a transverse electric field: Tight binding approach

Science.gov (United States)

Chegel, Raad; Behzad, Somayeh

2013-11-01

We have investigated the electronic properties of SiNTs, under the external electric field, using Tight Binding (TB) approximation. It was found that the energy levels, energy gaps, and density of states (DOS) strongly depend on the electric field strength. The large electric strength leads to coupling the neighbor subbands and induce destruction of subband degeneracy, increase of low-energy states, and strong modulation of energy gap which these effects reflect in the DOS spectrum. It has been shown that, the band gap reduction of Si g-NTs is linearly proportional to the electric field strength. The band gap variation for Si h-NTs increases first and later decreases (Metallic) or first remains constant and then decreases (semiconductor). Also we show that the larger diameter tubes are more sensitive to the field strength than smaller ones. The semiconducting metallic transition or vice versa can be achieved through an increasing of applied fields. Number and position of peaks in DOS spectrum are dependent on electric field strength.

PASTA in Penicillin Binding Proteins and Serine/Threonine Kinases: A Recipe of Structural, Dynamic and Binding Properties.

Science.gov (United States)

Calvanese, Luisa; Falcigno, Lucia; Squeglia, Flavia; D'Auria, Gabriella; Berisio, Rita

2017-11-24

Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs. Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles. Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Les faits, une arme contre la pauvreté

International Development Research Centre (IDRC) Digital Library (Canada)

L'engagement politique est essentiel à la viabilité du CBMS. .... La stratégie de mise en oeuvre du CBMS, qui consiste à faire participer les collectivités ...... travailleurs, de promouvoir l'emploi et de mettre sur pied des foires de recrutement.

FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription

International Nuclear Information System (INIS)

Holmqvist, Per-Henrik; Belikov, Sergey; Zaret, Kenneth S.; Wrange, Oerjan

2005-01-01

Novel binding sites for the forkhead transcription factor family member Forkhead box A (FoxA), previously referred to as Hepatocyte Nuclear Factor 3 (HNF3), were found within the mouse mammary tumor virus long terminal repeat (MMTV LTR). The effect of FoxA1 on MMTV LTR chromatin structure, and expression was evaluated in Xenopus laevis oocytes. Mutagenesis of either of the two main FoxA binding sites showed that the distal site, -232/-221, conferred FoxA1-dependent partial inhibition of glucocorticoid receptor (GR) driven MMTV transcription. The proximal FoxA binding segment consisted of two individual FoxA sites at -57/-46 and -45/-34, respectively, that mediated an increased basal MMTV transcription. FoxA1 binding altered the chromatin structure of both the inactive- and the hormone-activated MMTV LTR. Hydroxyl radical foot printing revealed FoxA1-mediated changes in the nucleosome arrangement. Micrococcal nuclease digestion showed the hormone-dependent sub-nucleosome complex, containing ∼120 bp of DNA, to be expanded by FoxA1 binding to the proximal segment into a larger complex containing ∼200 bp. The potential function of the FoxA1-mediated expression of the MMTV provirus for maintenance of expression in different tissues is discussed

Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

Science.gov (United States)

Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

2011-02-01

Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

Science.gov (United States)

Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

2015-03-27

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

On the influence of reward on action-effect binding

Directory of Open Access Journals (Sweden)

Paul Simon Muhle-Karbe

2012-11-01

Full Text Available Ideomotor theory states that the formation of anticipatory representations about the perceptual consequences of an action (i.e. action-effect (A-E binding provides the functional basis of voluntary action control. A host of studies has demonstrated that A-E binding occurs fast and effortlessly, yet only little is known about cognitive and affective factors that influence this learning process. In the present study, we sought to test whether the motivational value of an action modulates the acquisition of A-E associations. To this end, we associated specific actions with monetary incentives during the acquisition of novel A-E mappings. In a subsequent test phase, the degree of binding was assessed by presenting the former effect stimuli as task-irrelevant response primes in a forced-choice response task in the absence of any reward. Binding, as indexed by response priming through the former action effects, was only found for reward-related A-E mappings. Moreover, the degree to which reward associations modulated the binding strength was predicted by individuals’ trait sensitivity to reward. These observations indicate that the association of actions and their immediate outcomes depends on the motivational value of the action during learning, as well as on the motivational disposition of the individual. On a larger scale, these findings also highlight the link between ideomotor theories and reinforcement-learning theories, providing an interesting perspective for future research on anticipatory regulation of behavior.

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

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Amanda Tse

Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

Science.gov (United States)

Tse, Amanda; Verkhivker, Gennady M.

2015-01-01

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

(/sup 14/C)-L-valine binding to membranes of the frontal cortex in hepatic encephalopathy

Energy Technology Data Exchange (ETDEWEB)

Kienzl, E.; Riederer, P.; Jellinger, K. (Krankenhaus der Stadt Wien-Lainz (Austria). Ludwig Boltzmann Inst. fuer Neurobiologie); Kleinberger, G. (Vienna Univ. (Austria). 1. Medizinische Klinik)

1982-01-01

Serotonin (5-HT) receptors are modulated by L-valine (VAL). For further characterization of this effect a binding assay of (/sup 14/C)-L-VAL has been developed. A brief description of the experimental conditions is given. Moreover, measurement of VAL-binding has been applied to human brain tissue either from controls or hepatic failure. A marked increase of VAL-binding sites with no change in affinity was noted in hepatic coma, while in patients treated with parenteral nutrition plus VAL no such change could be measured. It is concluded that the beneficial therapeutic effects of VAL in hepatic encephalopathy are, at least in part, due to its modulating action on postsynaptic receptor membranes.

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

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Thijs Roebroek

2017-09-01

Full Text Available Reversibly switchable fluorescent proteins (RSFPs enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties.

A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

DEFF Research Database (Denmark)

Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise

2013-01-01

We present a full-length a1b2c2 GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate......-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode...... of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines a1T206 and c2T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important a1H101 and the N-methyl group near a1Y159, a1T206, and a1Y209. We present a binding mode...

Circular Business Model Challenges and Lessons Learned—An Industrial Perspective

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Pejvak Oghazi

2018-03-01

Full Text Available Both practitioners and researchers are concerned about resource deficiencies on the planet earth and agree that circular business models (CBMs represent solutions to move towards zero waste, improving environmental impacts and increasing economic profit. Despite all of the benefits of CBMs, the implications are not widely available, and failure rates are high. Thus, there is a need to identify the obstacles that stand in the way of CBM transition. This paper aims to identify the primary challenges of CBMs. Multiple case studies are employed, incorporating six companies and data gleaned from 17 in-depth interviews. Theoretical and managerial implications are described at the end of the study.

Expression of the C-terminal family 22 carbohydratebinding module ...

African Journals Online (AJOL)

... not reveal marked cell wall phenotype. In addition, there were no observable changes in the height or the appearance of the transgenic plants expressing the CBM22-2 module. The results indicate that the family 22 carbohydrate binding module is not a potential candidate for use in in planta modification of the cell wall.

Prediction of tissue-specific cis-regulatory modules using Bayesian networks and regression trees

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Chen Xiaoyu

2007-12-01

Full Text Available Abstract Background In vertebrates, a large part of gene transcriptional regulation is operated by cis-regulatory modules. These modules are believed to be regulating much of the tissue-specificity of gene expression. Results We develop a Bayesian network approach for identifying cis-regulatory modules likely to regulate tissue-specific expression. The network integrates predicted transcription factor binding site information, transcription factor expression data, and target gene expression data. At its core is a regression tree modeling the effect of combinations of transcription factors bound to a module. A new unsupervised EM-like algorithm is developed to learn the parameters of the network, including the regression tree structure. Conclusion Our approach is shown to accurately identify known human liver and erythroid-specific modules. When applied to the prediction of tissue-specific modules in 10 different tissues, the network predicts a number of important transcription factor combinations whose concerted binding is associated to specific expression.

A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

Energy Technology Data Exchange (ETDEWEB)

Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M. (VA); (UCLA); (Vanderbilt); (UCSF)

2014-10-02

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

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Romain Pardoux

Full Text Available To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9TKE(12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d = 25±6 nM to K(d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d = 0.25±0.06 nM. FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as(P-O and ν(s(P-O IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as(UO(2(2+ vibration (from 923 cm(-1 to 908 cm(-1 was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

Modulating uranium binding affinity in engineered Calmodulin EF-hand peptides: effect of phosphorylation

International Nuclear Information System (INIS)

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Guilloreau, Luc; Berthomieu, Catherine; Delangle, Pascale; Adriano, Jean-Marc

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T 9 TKE 12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K d =25±6 nM to K d =5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the sub-nanomolar range (K d = 0.25±0.06 nM). FTIR analyses showed that the phospho-threonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν as (P-O) and ν s (P-O) IR modes of phospho-threonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν as (UO 2 ) 2+ vibration (from 923 cm -1 to 908 cm -1 ) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. (authors)

The starch-binding domain family CBM41 - an in silico analysis of evolutionary relationships

DEFF Research Database (Denmark)

Janeček, Štefan; Majzlová, Katarína; Svensson, Birte

2017-01-01

Within the CAZy database, there are 81 carbohydrate-binding module (CBM) families. A CBM represents a non-catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch-binding domains from the family CBM41 that are usually part...

LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

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Lisnyak Yu. V.

2017-10-01

Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

High throughput techniques for discovering new glycine receptor modulators and their binding sites

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Daniel F Gilbert

2009-10-01

Full Text Available The inhibitory glycine receptor (GlyR is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific compounds. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384 well plate using nanogram quantities of cDNA.

Positive modulator of bone morphogenic protein-2

Science.gov (United States)

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY; Takahashi, Kazuyuki [Germantown, MD

2009-01-27

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

Positive modulator of bone morphogenic protein-2

Energy Technology Data Exchange (ETDEWEB)

Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Kazuyuki, Takahashi

2017-06-06

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

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.

Dissociation of binding and learning processes.

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Moeller, Birte; Frings, Christian

2017-11-01

A single encounter of a stimulus together with a response can result in a short-lived association between the stimulus and the response [sometimes called an event file, see Hommel, Müsseler, Aschersleben, & Prinz, (2001) Behavioral and Brain Sciences, 24, 910-926]. The repetition of stimulus-response pairings typically results in longer lasting learning effects indicating stimulus-response associations (e.g., Logan & Etherton, (1994) Journal of Experimental Psychology: Learning, Memory, and Cognition, 20, 1022-1050]. An important question is whether or not what has been described as stimulus-response binding in action control research is actually identical with an early stage of incidental learning (e.g., binding might be seen as single-trial learning). Here, we present evidence that short-lived binding effects can be distinguished from learning of longer lasting stimulus-response associations. In two experiments, participants always responded to centrally presented target letters that were flanked by response irrelevant distractor letters. Experiment 1 varied whether distractors flanked targets on the horizontal or vertical axis. Binding effects were larger for a horizontal than for a vertical distractor-target configuration, while stimulus configuration did not influence incidental learning of longer lasting stimulus-response associations. In Experiment 2, the duration of the interval between response n - 1 and presentation of display n (500 ms vs. 2000 ms) had opposing influences on binding and learning effects. Both experiments indicate that modulating factors influence stimulus-response binding and incidental learning effects in different ways. We conclude that distinct underlying processes should be assumed for binding and incidental learning effects.

Acetylcholinesterase potentiates [{sup 3}H]fluorowillardiine and [{sup 3}H]AMPA binding to rat cortical membranes

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Olivera, S.; Rodriguez-Ithurralde, D. [Department of Anatomy, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD (United Kingdom); Henley, J.M. [Molecular Neuroscience Unit, Division Neuromyology, Instituto de Investigaciones Biologicas Clemente Estable, 11600 Montevideo (Uruguay)

1999-04-01

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[{sup 3}H]5-fluorowillardiine ([{sup 3}H]FW) and [{sup 3}H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to {approx}60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [{sup 3}H]kainate binding. [{sup 3}H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC{sub 50} values of 4x10{sup -5} and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [{sup 3}H]FW binding is Ca{sup 2+}- and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [{sup 3}H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the B{sub max} of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca²⁺ channels

DEFF Research Database (Denmark)

Garau, Gianpiero; Magotti, Paola; Heine, Martin

2015-01-01

Our previous studies revealed that L-type voltage-dependent Ca2+ channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular......M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.......2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing...

Military confidence building on the Korean Peninsula: possible first steps toward cooperation

Energy Technology Data Exchange (ETDEWEB)

Vannoni, M.

1997-05-01

The Korean Peninsula is one of the world`s most tense military confrontational sites. Nearly 2 million North Korean, South Korean, and U.S. troops face each other along the 255-km long military demarcation line. Confidence building measures (CBMs), particularly military ones, that address the security needs of both countries could decrease the danger of conflict and help create an environment where a peace regime might be negotiated. In spite of the present high level of mutual distrust, steps can still be taken to prepare for future development and implementation of CBMs. This paper defines some simple and specific first steps toward CBMs that might be useful on the Korean Peninsula.

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process

Science.gov (United States)

Reshetnikov, Roman V.; Sponer, Jiri; Rassokhina, Olga I.; Kopylov, Alexei M.; Tsvetkov, Philipp O.; Makarov, Alexander A.; Golovin, Andrey V.

2011-01-01

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange. PMID:21893589

Integrin-directed modulation of macrophage responses to biomaterials.

Science.gov (United States)

Zaveri, Toral D; Lewis, Jamal S; Dolgova, Natalia V; Clare-Salzler, Michael J; Keselowsky, Benjamin G

2014-04-01

Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials. Copyright © 2014 Elsevier Ltd. All rights reserved.

Localization of 125I-insulin binding sites in the rat hypothalamus by quantitative autoradiography

International Nuclear Information System (INIS)

Corp, E.S.; Woods, S.C.; Figlewicz, D.P.; Porte, D. Jr.; Baskin, D.G.; Dorsa, D.M.

1986-01-01

In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125 I-insulin in the hypothalamus of the rat brain. Highest specific binding was found in the arculate, dorsomedial, suprachiasmatic, paraventricular and periventricular regions. Significantly lower binding was present in the ventromedial nucleus and median eminence. The results are consistent with the hypothesis that insulin modulates the neural regulation of feeding by acting at sites in the hypothalamus. (author)

The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

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John M Morrison

2012-03-01

Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

Fatty acid modulated human serum albumin binding of the β-carboline alkaloids norharmane and harmane.

Science.gov (United States)

Domonkos, Celesztina; Fitos, Ilona; Visy, Júlia; Zsila, Ferenc

2013-12-02

Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA.

Optical binding of two microparticles levitated in vacuum

Science.gov (United States)

Arita, Yoshihiko; Wright, Ewan M.; Dholakia, Kishan

2017-04-01

Optical binding refers to an optically mediated inter-particle interaction that creates new equilibrium positions for closely spaced particles [1-5]. Optical binding of mesoscopic particles levitated in vacuum can pave the way towards the realisation of a large scale quantum bound array in cavity-optomechanics [6-9]. Recently we have demonstrated trapping and rotation of two mesoscopic particles in vacuum using a spatial-light-modulator-based approach to trap more than one particle, induce controlled rotation of individual particles, and mediate interparticle separation [10]. By trapping and rotating two vaterite particles, we observe intensity modulation of the scattered light at the sum and difference frequencies with respect to the individual rotation rates. This first demonstration of optical interference between two microparticles in vacuum has lead to a platform to explore optical binding. Here we demonstrate for the first time optically bound two microparticles mediated by light scattering in vacuum. We investigate autocorrelations between the two normal modes of oscillation, which are determined by the centre-of-mass and the relative positions of the two-particle system. In situ determination of the optical restoring force acting on the bound particles are based on measurement of the oscillation frequencies of the autocorrelation functions of the two normal modes, thereby providing a powerful and original platform to explore multiparticle entanglement in cavity-optomechanics.

Identification of a polyoxometalate inhibitor of the DNA binding activity of Sox2.

Science.gov (United States)

Narasimhan, Kamesh; Pillay, Shubhadra; Bin Ahmad, Nor Rizal; Bikadi, Zsolt; Hazai, Eszter; Yan, Li; Kolatkar, Prasanna R; Pervushin, Konstantin; Jauch, Ralf

2011-06-17

Aberrant expression of transcription factors is a frequent cause of disease, yet drugs that modulate transcription factor protein-DNA interactions are presently unavailable. To this end, the chemical tractability of the DNA binding domain of the stem cell inducer and oncogene Sox2 was explored in a high-throughput fluorescence anisotropy screen. The screening revealed a Dawson polyoxometalate (K(6)[P(2)Mo(18)O(62)]) as a direct and nanomolar inhibitor of the DNA binding activity of Sox2. The Dawson polyoxometalate (Dawson-POM) was found to be selective for Sox2 and related Sox-HMG family members when compared to unrelated paired and zinc finger DNA binding domains. [(15)N,(1)H]-Transverse relaxation optimized spectroscopy (TROSY) experiments coupled with docking studies suggest an interaction site of the POM on the Sox2 surface that enabled the rationalization of its inhibitory activity. The unconventional molecular scaffold of the Dawson-POM and its inhibitory mode provides strategies for the development of drugs that modulate transcription factors.

CCL2 binding is CCR2 independent in primary adult human astrocytes.

Science.gov (United States)

Fouillet, A; Mawson, J; Suliman, O; Sharrack, B; Romero, I A; Woodroofe, M N

2012-02-09

Chemokines are low relative molecular mass proteins, which have chemoattractant actions on many cell types. The chemokine, CCL2, has been shown to play a major role in the recruitment of monocytes in central nervous system (CNS) lesions in multiple sclerosis (MS). Since resident astrocytes constitute a major source of chemokine synthesis including CCL2, we were interested to assess the regulation of CCL2 by astrocytes. We showed that CCL2 bound to the cell surface of astrocytes and binding was not modulated by inflammatory conditions. However, CCR2 protein was not detected nor was activation of the classical CCR2 downstream signaling pathways. Recent studies have shown that non-signaling decoy chemokine receptors bind and modulate the expression of chemokines at site of inflammation. Here, we show that the D6 chemokine decoy receptor is constitutively expressed by primary human adult astrocytes at both mRNA and protein level. In addition, CCL3, which binds to D6, but not CCL19, which does not bind to D6, displaced CCL2 binding to astrocytes; indicating that CCL2 may bind to this cell type via the D6 receptor. Our results suggest that CCL2 binding to primary adult human astrocytes is CCR2-independent and is likely to be mediated via the D6 decoy chemokine receptor. Therefore we propose that astrocytes are implicated in both the establishment of chemokine gradients for the migration of leukocytes into and within the CNS and in the regulation of CCL2 levels at inflammatory sites in the CNS. Copyright © 2011 Elsevier B.V. All rights reserved.

Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta selective ligand binding.

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Fernanda A H Batista

Full Text Available Peroxisome proliferator activated receptors (PPARs δ, α and γ are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328 in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

Identification of an allosteric binding site for RORγt inhibition

Energy Technology Data Exchange (ETDEWEB)

Scheepstra, Marcel; Leysen, Seppe; vanAlmen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc (Merck); (Eindhoven)

2015-12-07

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains

Science.gov (United States)

Cong, Le; Zhou, Ruhong; Kuo, Yu-chi; Cunniff, Margaret; Zhang, Feng

2012-01-01

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs. PMID:22828628

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

Science.gov (United States)

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

Impact of load-related neural processes on feature binding in visuospatial working memory.

Directory of Open Access Journals (Sweden)

Nicole A Kochan

Full Text Available BACKGROUND: The capacity of visual working memory (WM is substantially limited and only a fraction of what we see is maintained as a temporary trace. The process of binding visual features has been proposed as an adaptive means of minimising information demands on WM. However the neural mechanisms underlying this process, and its modulation by task and load effects, are not well understood. OBJECTIVE: To investigate the neural correlates of feature binding and its modulation by WM load during the sequential phases of encoding, maintenance and retrieval. METHODS AND FINDINGS: 18 young healthy participants performed a visuospatial WM task with independent factors of load and feature conjunction (object identity and position in an event-related functional MRI study. During stimulus encoding, load-invariant conjunction-related activity was observed in left prefrontal cortex and left hippocampus. During maintenance, greater activity for task demands of feature conjunction versus single features, and for increased load was observed in left-sided regions of the superior occipital cortex, precuneus and superior frontal cortex. Where these effects were expressed in overlapping cortical regions, their combined effect was additive. During retrieval, however, an interaction of load and feature conjunction was observed. This modulation of feature conjunction activity under increased load was expressed through greater deactivation in medial structures identified as part of the default mode network. CONCLUSIONS AND SIGNIFICANCE: The relationship between memory load and feature binding qualitatively differed through each phase of the WM task. Of particular interest was the interaction of these factors observed within regions of the default mode network during retrieval which we interpret as suggesting that at low loads, binding processes may be 'automatic' but at higher loads it becomes a resource-intensive process leading to disengagement of activity in this

QSAR of estrogen receptor modulators: exploring selectivity requirements for ER(alpha) versus ER(beta) binding of tetrahydroisoquinoline derivatives using E-state and physicochemical parameters.

Science.gov (United States)

Mukherjee, Subhendu; Saha, Achintya; Roy, Kunal

2005-02-15

Considering importance of developing selective estrogen receptor modulators (SERMs), the present paper explores selectivity requirements of tetrahydroisoquinoline derivatives for binding with ER(alpha) versus ER(beta) receptors using E-state index and physicochemical parameters. The best model [n=21, Q(2)=0.512, R(a)(2)=0.613, R=0.819, F=11.6 (df 3,17)] for ER(alpha) binding data obtained from radioligand binding assay showed importance of C(1), C(15) and lipophilicity (logP) while the best model [n=21, Q(2)=0.768, R(a)(2)=0.796, R=0.904, F=40.1 (df 2,18)] for ER(beta) binding data showed importance of C(1) and molar refractivity (MR). While modeling ER(alpha)/ER(beta) selectivity [n=21, Q(2)=0.695, R(a)(2)=0.739, R=0.882, F=19.8 (df 3,17)], C(1), C(15) and molar refractivity were found to be significant contributors. The data obtained from cellular transcription assay were also modeled. In case of ER(alpha), the best equation involving E-state values of C(1) and C(14) and logP explained 62.1% of the variance while the best equation for ER(beta) involving E-state values of C(1) and C(15) and MR explained 64.6% of the variance of the response variable. In case of ER(alpha)/ER(beta) selectivity, the best equation involving E-state values of O(8), C(14) and N(27) showed 48.3% explained variance, which increased to 63.5% on deletion of single outlier. From the analysis it appears that the nitrogen atom of the aminoethoxyphenyl substituent and 6-hydroxy substituent of the tetrahydroisoquinoline nucleus play important roles for ER(alpha)/ER(beta) selectivity in addition to R(1) and R(2) substituents.

Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation

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Henrissat Bernard

2011-09-01

Full Text Available Abstract Background Spore-forming Bacilli are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI-tract of animals. Isolates of various Bacillus species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms. Results We report the annotation of carbohydrate active enzymes (CAZymes of two pigmented Bacilli isolated from the human GI-tract and belonging to the Bacillus indicus and B. firmus species. A high number of glycoside hydrolases (GHs and carbohydrate binding modules (CBMs were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both Bacilli. Experimental results on biofilm formation confirmed genomic data on the potentials of B. indicus HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both Bacilli to degrade mammalian glycans. Conclusions CAZy analyses of the genomes of the two pigmented Bacilli, compared to other Bacillus species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented Bacilli are adapted to the intestinal environment and are suited to grow in and colonize the human gut.

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process.

Science.gov (United States)

Reshetnikov, Roman V; Sponer, Jiri; Rassokhina, Olga I; Kopylov, Alexei M; Tsvetkov, Philipp O; Makarov, Alexander A; Golovin, Andrey V

2011-12-01

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange. © The Author(s) 2011. Published by Oxford University Press.

Reconstitution of high-affinity opioid agonist binding in brain membranes

Energy Technology Data Exchange (ETDEWEB)

Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

1991-03-15

In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

Biochemical profiling of histone binding selectivity of the yeast bromodomain family.

Directory of Open Access Journals (Sweden)

Qiang Zhang

2010-01-01

Full Text Available It has been shown that molecular interactions between site-specific chemical modifications such as acetylation and methylation on DNA-packing histones and conserved structural modules present in transcriptional proteins are closely associated with chromatin structural changes and gene activation. Unlike methyl-lysine that can interact with different protein modules including chromodomains, Tudor and MBT domains, as well as PHD fingers, acetyl-lysine (Kac is known thus far to be recognized only by bromodomains. While histone lysine acetylation plays a crucial role in regulation of chromatin-mediated gene transcription, a high degree of sequence variation of the acetyl-lysine binding site in the bromodomains has limited our understanding of histone binding selectivity of the bromodomain family. Here, we report a systematic family-wide analysis of 14 yeast bromodomains binding to 32 lysine-acetylated peptides derived from known major acetylation sites in four core histones that are conserved in eukaryotes.The histone binding selectivity of purified recombinant yeast bromodomains was assessed by using the native core histones in an overlay assay, as well as N-terminally biotinylated lysine-acetylated histone peptides spotted on streptavidin-coated nitrocellulose membrane in a dot blot assay. NMR binding analysis further validated the interactions between histones and selected bromodomain. Structural models of all yeast bromodomains were built using comparative modeling to provide insights into the molecular basis of their histone binding selectivity.Our study reveals that while not all members of the bromodomain family are privileged to interact with acetylated-lysine, identifiable sequence features from those that bind histone emerge. These include an asparagine residue at the C-terminus of the third helix in the 4-helix bundle, negatively charged residues around the ZA loop, and preponderance of aromatic amino acid residues in the binding pocket

Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

Science.gov (United States)

Kamina, Anyango D; Williams, Noreen

2017-01-01

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

Integration of the Shuttle RMS/CBM Positioning Virtual Environment Simulation

Science.gov (United States)

Dumas, Joseph D.

1996-01-01

Constructing the International Space Station, or other structures, in space presents a number of problems. In particular, payload restrictions for the Space Shuttle and other launch mechanisms prohibit assembly of large space-based structures on Earth. Instead, a number of smaller modules must be boosted into orbit separately and then assembled to form the final structure. The assembly process is difficult, as docking interfaces such as Common Berthing Mechanisms (CBMS) must be precisely positioned relative to each other to be within the "capture envelope" (approximately +/- 1 inch and +/- 0.3 degrees from the nominal position) and attach properly. In the case of the Space Station, the docking mechanisms are to be positioned robotically by an astronaut using the 55-foot-long Remote Manipulator System (RMS) robot arm. Unfortunately, direct visual or video observation of the placement process is difficult or impossible in many scenarios. One method that has been tested for aligning the CBMs uses a boresighted camera mounted on one CBM to view a standard target on the opposing CBM. While this method might be sufficient to achieve proper positioning with considerable effort, it does not provide a high level of confidence that the mechanisms have been placed within capture range of each other. It also does nothing to address the risk of inadvertent contact between the CBMS, which could result in RMS control software errors. In general, constraining the operator to a single viewpoint with few, if any, depth cues makes the task much more difficult than it would be if the target could be viewed in three-dimensional space from various viewpoints. The actual work area could be viewed by an astronaut during EVA; however, it would be extremely impractical to have an astronaut control the RMS while spacewalking. On the other hand, a view of the RMS and CBMs to be positioned in a virtual environment aboard the Space Shuttle orbiter or Space Station could provide similar benefits

Effect of anticonvulsant drugs on (/sup 35/S)t-butylbicyclophosphorothionate binding in vitro and ex vivo

Energy Technology Data Exchange (ETDEWEB)

Pitkaenen, A.; Riekkinen, P.J.; Saano, V.; Tuomisto, L.

1987-01-01

Using several concentrations of eight anticonvulsant drugs in clinical use (carbamazepine, clonazepam, phenytoin, phenobarbital, ethosuximide, primidone, sodium valproate, and D,L-..gamma..-vinyl GABA), we studied their abilities in vitro to displace (/sup 35/S)t-butylbicyclophosphorothionate (/sup 35/S-TBPS) from its binding site in a homogenate of rat brain. Thereafter ethosuximide (150 mg/kg), phenobarbital (30 mg/kg), clonazepam (0.3 mg/kg), or phenytoin (100 mg/kg) was injected intraperitoneally into rats for 16-20 days; and the effect of drug administration on /sup 35/S-TBPS binding was studied in the cortex and hippocampus ex vivo. Phenobarbital (100 ..mu..M, P<0.001), ethosuximide (500 ..mu..M, P<0.001), and phenytoin (40 ..mu..M, P<0.001) decreased the specific /sup 35/S-TBPS binding in vitro by 10-16%. After drug administration of phenobarbital (concentration in plasma 168 ..mu..M), the number of binding sites decreased and the binding affinity (p<0.05) in the cortex increased. Other anticonvulsants did not modulate /sup 35/S-TBPS binding in vitro at the concentration analogous to therapeutic plasma levels or ex vivo at the dose used. These results suggest that the use of phenobarbital may modulate the TBPS binding site, but the role of the present findings in the anticonvulsant action of phenobarbital needs to be further studied.

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

Science.gov (United States)

Duan, Cheng-Jie; Huang, Ming-Yue; Pang, Hao; Zhao, Jing; Wu, Chao-Xing; Feng, Jia-Xun

2017-07-01

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat /K M ) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

Reflection-Based Python-C++ Bindings

International Nuclear Information System (INIS)

Generowicz, Jacek; Lavrijsen, Wim T.L.P.; Marino, Massimo; Mato, Pere

2004-01-01

Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called extension modules. A number of tools for easing the process of creating such bindings exist, such as SWIG and Boost. Python. Yet, the process still requires a considerable amount of effort and expertise. The C++ language has few built-in introspective capabilities, but tools such as LCGDict and CINT add this by providing so-called dictionaries: libraries that contain information about the names, entry points, argument types, etc. of other libraries. The reflection information from these dictionaries can be used for the creation of bindings and so the process can be fully automated, as dictionaries are already provided for many end-user libraries for other purposes, such as object persistency. PyLCGDict is a Python extension module that uses LCG dictionaries, as PyROOT uses CINT reflection information, to allow /cwPython users to access C++ libraries with essentially no preparation on the users' behalf. In addition, and in a similar way, PyROOT gives ROOT users access to Python libraries

Interferon Lambda: Modulating Immunity in Infectious Diseases.

Science.gov (United States)

Syedbasha, Mohammedyaseen; Egli, Adrian

2017-01-01

Interferon lambdas (IFN-λs; IFNL1-4) modulate immunity in the context of infections and autoimmune diseases, through a network of induced genes. IFN-λs act by binding to the heterodimeric IFN-λ receptor (IFNLR), activating a STAT phosphorylation-dependent signaling cascade. Thereby hundreds of IFN-stimulated genes are induced, which modulate various immune functions via complex forward and feedback loops. When compared to the well-characterized IFN-α signaling cascade, three important differences have been discovered. First, the IFNLR is not ubiquitously expressed: in particular, immune cells show significant variation in the expression levels of and susceptibilities to IFN-λs. Second, the binding affinities of individual IFN-λs to the IFNLR varies greatly and are generally lower compared to the binding affinities of IFN-α to its receptor. Finally, genetic variation in the form of a series of single-nucleotide polymorphisms (SNPs) linked to genes involved in the IFN-λ signaling cascade has been described and associated with the clinical course and treatment outcomes of hepatitis B and C virus infection. The clinical impact of IFN-λ signaling and the SNP variations may, however, reach far beyond viral hepatitis. Recent publications show important roles for IFN-λs in a broad range of viral infections such as human T-cell leukemia type-1 virus, rotaviruses, and influenza virus. IFN-λ also potentially modulates the course of bacterial colonization and infections as shown for Staphylococcus aureus and Mycobacterium tuberculosis . Although the immunological processes involved in controlling viral and bacterial infections are distinct, IFN-λs may interfere at various levels: as an innate immune cytokine with direct antiviral effects; or as a modulator of IFN-α-induced signaling via the suppressor of cytokine signaling 1 and the ubiquitin-specific peptidase 18 inhibitory feedback loops. In addition, the modulation of adaptive immune functions via macrophage

One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR-dependent hemostatic activity in vivo.

Science.gov (United States)

Pavani, G; Zintner, S M; Ivanciu, L; Small, J C; Stafford, K A; Szeto, J H; Margaritis, P

2017-03-01

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo. Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in

Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

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Olgun eGuvench

2015-06-01

Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

Down-regulation of endothelin binding sites in rat vascular smooth muscle cells

International Nuclear Information System (INIS)

Roubert, P.; Gillard, V.; Plas, P.; Chabrier, P.E.; Braquet, P.

1990-01-01

In cultured rat aortic smooth muscle cells, [ 125 I]endothelin (ET-1) bound to an apparent single class of high affinity recognition sites with a dissociation constant of 1.84 +/- 0.29 nmol/L and a maximum binding of 62 +/- 10.5 fmol/10(6) cells. The binding was not affected by calcium antagonists or vasoactive substances, including angiotensin II, arginine vasopressin, atrial natriuretic factor and bradykinin. Exposure of the cells to ET-1 (0.01 nmol/L to 10 nmol/L) resulted in an apparent dose-dependent reduction of the number of endothelin binding sites with no significant modification of its binding affinity. The time course of the down-regulation of ET-1 binding sites showed that this effect was present after 30 min incubation and persisted after 18 h. This indicates that down-regulation of ET-1 binding sites can modulate the activity of ET-1 and suggests a rapid internalization of ET-1 in vascular cells

The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

DEFF Research Database (Denmark)

Morrill, Johan; Kulcinskaja, Evelina; Sulewska, Anna Maria

2015-01-01

β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here....... Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs...

Transfer-matrix approach for modulated structures with defects

International Nuclear Information System (INIS)

Kostyrko, T.

2000-01-01

We consider scattering of electrons by defects in a periodically modulated, quasi-one-dimensional structure, within a tight-binding model. Combining a transfer matrix method and a Green function method we derive a formula for a Landauer conductance and show its equivalence to the result of Kubo linear response theory. We obtain explicitly unperturbed lattice Green functions from their equations of motion, using the transfer matrices. We apply the presented formalism in computations of the conductance of several multiband modulated structures with defects: (a) carbon nanotubes (b) two-dimensional (2D) superlattice (c) modulated leads with 1D wire in the tunneling regime. (c) 2000 The American Physical Society

Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

International Nuclear Information System (INIS)

Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

1987-01-01

The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

Tunable GLUT-Hexose Binding and Transport via Modulation of Hexose C-3 Hydrogen-Bonding Capabilities.

Science.gov (United States)

Kumar Kondapi, Venkata Pavan; Soueidan, Olivier-Mohamad; Cheeseman, Christopher I; West, Frederick G

2017-06-12

The importance of the hydrogen bonding interactions in the GLUT-hexose binding process (GLUT=hexose transporter) has been demonstrated by studying the binding of structurally modified d-fructose analogues to GLUTs, and in one case its transport into cells. The presence of a hydrogen bond donor at the C-3 position of 2,5-anhydro-d-mannitol derivatives is essential for effective binding to GLUT5 and transport into tumor cells. Surprisingly, installation of a group that can function only as a hydrogen bond acceptor at C-3 resulted in selective recognition by GLUT1 rather than GLUT5. A fluorescently labelled analogue clearly showed GLUT-mediated transport and low efflux properties of the probe. This study reveals that a single positional modification of a 2,5-anhydro-d-mannitol derivative is sufficient to switch its binding preference from GLUT5 to GLUT1, and uncovers general scaffolds that are suitable for the potential selective delivery of molecular payloads into tumor cells via GLUT transport machinery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative structural analysis of lipid binding START domains.

Directory of Open Access Journals (Sweden)

Ann-Gerd Thorsell

Full Text Available Steroidogenic acute regulatory (StAR protein related lipid transfer (START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

Science.gov (United States)

Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

2009-10-01

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Mycobacterium smegmatis Ku binds DNA without free ends.

Science.gov (United States)

Kushwaha, Ambuj K; Grove, Anne

2013-12-01

Ku is central to the non-homologous end-joining pathway of double-strand-break repair in all three major domains of life, with eukaryotic homologues being associated with more diversified roles compared with prokaryotic and archaeal homologues. Ku has a conserved central 'ring-shaped' core domain. While prokaryotic homologues lack the N- and C-terminal domains that impart functional diversity to eukaryotic Ku, analyses of Ku from certain prokaryotes such as Pseudomonas aeruginosa and Mycobacterium smegmatis have revealed the presence of distinct C-terminal extensions that modulate DNA-binding properties. We report in the present paper that the lysine-rich C-terminal extension of M. smegmatis Ku contacts the core protein domain as evidenced by an increase in DNA-binding affinity and a decrease in thermal stability and intrinsic tryptophan fluorescence upon its deletion. Ku deleted for this C-terminus requires free DNA ends for binding, but translocates to internal DNA sites. In contrast, full-length Ku can directly bind DNA without free ends, suggesting that this property is conferred by its C-terminus. Such binding to internal DNA sites may facilitate recruitment to sites of DNA damage. The results of the present study also suggest that extensions beyond the shared core domain may have independently evolved to expand Ku function.

Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.

Science.gov (United States)

Yamasaki, Takashi; Nakazaki, Yosuke; Yoshida, Masasuke; Watanabe, Yo-hei

2011-07-01

ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer. © 2011 The Authors Journal compilation © 2011 FEBS.

Water-Hydrogel Binding Affinity Modulates Freeze-Drying-Induced Micropore Architecture and Skeletal Myotube Formation.

Science.gov (United States)

Rich, Max H; Lee, Min Kyung; Marshall, Nicholas; Clay, Nicholas; Chen, Jinrong; Mahmassani, Ziad; Boppart, Marni; Kong, Hyunjoon

2015-08-10

Freeze-dried hydrogels are increasingly used to create 3D interconnected micropores that facilitate biomolecular and cellular transports. However, freeze-drying is often plagued by variance in micropore architecture based on polymer choice. We hypothesized that water-polymer binding affinity plays a significant role in sizes and numbers of micropores formed through freeze-drying, influencing cell-derived tissue quality. Poly(ethylene glycol)diacrylate (PEGDA) hydrogels with alginate methacrylate (AM) were used due to AM's higher binding affinity for water than PEGDA. PEGDA-AM hydrogels with larger AM concentrations resulted in larger sizes and numbers of micropores than pure PEGDA hydrogels, attributed to the increased mass of water binding to the PEGDA-AM gel. Skeletal myoblasts loaded in microporous PEGDA-AM hydrogels were active to produce 3D muscle-like tissue, while those loaded in pure PEGDA gels were localized on the gel surface. We propose that this study will be broadly useful in designing and improving the performance of various microporous gels.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

Science.gov (United States)

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).

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Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav; Daugvilaite, Viktorija; Steen, Anne; Brvar, Matjaž; Pui, Aurel; Frimurer, Thomas Michael; Ulven, Trond; Rosenkilde, Mette Marie

2016-12-23

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn 2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn 2+ is anchored to the chemokine receptor-conserved Glu-283 VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248 VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116 III:16/3.40 , a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125 I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37 I:07/1.39 , Trp-86 II:20/2.60 , and Phe-109 III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Loss of selenium-binding protein 1 decreases sensitivity to clastogens and intracellular selenium content in HeLa cells

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Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesized that loss of SBP1 modulates cellular selenium content and the response of ...

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

KIRMES: kernel-based identification of regulatory modules in euchromatic sequences.

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Schultheiss, Sebastian J; Busch, Wolfgang; Lohmann, Jan U; Kohlbacher, Oliver; Rätsch, Gunnar

2009-08-15

Understanding transcriptional regulation is one of the main challenges in computational biology. An important problem is the identification of transcription factor (TF) binding sites in promoter regions of potential TF target genes. It is typically approached by position weight matrix-based motif identification algorithms using Gibbs sampling, or heuristics to extend seed oligos. Such algorithms succeed in identifying single, relatively well-conserved binding sites, but tend to fail when it comes to the identification of combinations of several degenerate binding sites, as those often found in cis-regulatory modules. We propose a new algorithm that combines the benefits of existing motif finding with the ones of support vector machines (SVMs) to find degenerate motifs in order to improve the modeling of regulatory modules. In experiments on microarray data from Arabidopsis thaliana, we were able to show that the newly developed strategy significantly improves the recognition of TF targets. The python source code (open source-licensed under GPL), the data for the experiments and a Galaxy-based web service are available at http://www.fml.mpg.de/raetsch/suppl/kirmes/.

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

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Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H P; Whisstock, James C; Baker, Edward N; Kreikemeyer, Bernd

2012-11-02

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

DEFF Research Database (Denmark)

Matzen, L E; Kristensen, S R; Kvetny, J

1991-01-01

The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high ...

Anion Binding Studies on Receptors Derived from the Indolo[2,3-a]carbazole Scaffold Having Different Binding Cavity Sizes

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Guzmán Sánchez

2014-07-01

Full Text Available The indolo[2,3-a]carbazole scaffold is a fused polyheteroaromatic system bearing two NH groups which suitably converge as hydrogen bond donor sites for the recognition of anions. A simple derivatisation of the indolocarbazole system at positions 1 and 10 with different functional groups, namely alcohols and amides, has contributed to modulate the anion binding selectivity and sensibility. A particularly good response has been obtained for the benzoate anion.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

Energy Technology Data Exchange (ETDEWEB)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui; Mansfield, Elisabeth; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Vinzant, Todd

2017-04-24

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific

Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

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Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

2008-01-01

Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft with a charact......Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft...... will address surface sites in both barley alpha-amylase 1 and in the related isozyme 2....

Rational steering of insulin binding specificity by intra-chain chemical crosslinking

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Viková, Jitka; Collinsová, Michaela; Kletvíková, Emília; Buděšínský, Miloš; Kaplan, Vojtěch; Žáková, Lenka; Veverka, Václav; Hexnerová, Rozálie; Aviñó, Roberto J. Tarazona; Straková, Jana; Selicharová, Irena; Vaněk, Václav; Wright, Daniel W.; Watson, Christopher J.; Turkenburg, Johan P.; Brzozowski, Andrzej M.; Jiráček, Jiří

2016-01-01

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.

A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

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Brian Krumm

Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

DEFF Research Database (Denmark)

Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

2016-01-01

Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

Kaiso Directs the Transcriptional Corepressor MTG16 to the Kaiso Binding Site in Target Promoters

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Barrett, Caitlyn W.; Smith, J. Joshua; Lu, Lauren C.; Markham, Nicholas; Stengel, Kristy R.; Short, Sarah P.; Zhang, Baolin; Hunt, Aubrey A.; Fingleton, Barbara M.; Carnahan, Robert H.; Engel, Michael E.; Chen, Xi; Beauchamp, R. Daniel; Wilson, Keith T.; Hiebert, Scott W.; Reynolds, Albert B.; Williams, Christopher S.

2012-01-01

Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the ApcMin mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co

Identification of potential small molecule allosteric modulator sites on IL-1R1 ectodomain using accelerated conformational sampling method.

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Chao-Yie Yang

Full Text Available The interleukin-1 receptor (IL-1R is the founding member of the interleukin 1 receptor family which activates innate immune response by its binding to cytokines. Reports showed dysregulation of cytokine production leads to aberrant immune cells activation which contributes to auto-inflammatory disorders and diseases. Current therapeutic strategies focus on utilizing antibodies or chimeric cytokine biologics. The large protein-protein interaction interface between cytokine receptor and cytokine poses a challenge in identifying binding sites for small molecule inhibitor development. Based on the significant conformational change of IL-1R type 1 (IL-1R1 ectodomain upon binding to different ligands observed in crystal structures, we hypothesized that transient small molecule binding sites may exist when IL-1R1 undergoes conformational transition and thus suitable for inhibitor development. Here, we employed accelerated molecular dynamics (MD simulation to efficiently sample conformational space of IL-1R1 ectodomain. Representative IL-1R1 ectodomain conformations determined from the hierarchy cluster analysis were analyzed by the SiteMap program which leads to identify small molecule binding sites at the protein-protein interaction interface and allosteric modulator locations. The cosolvent mapping analysis using phenol as the probe molecule further confirms the allosteric modulator site as a binding hotspot. Eight highest ranked fragment molecules identified from in silico screening at the modulator site were evaluated by MD simulations. Four of them restricted the IL-1R1 dynamical motion to inactive conformational space. The strategy from this study, subject to in vitro experimental validation, can be useful to identify small molecule compounds targeting the allosteric modulator sites of IL-1R and prevent IL-1R from binding to cytokine by trapping IL-1R in inactive conformations.

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei.

Science.gov (United States)

Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J

2016-02-01

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC(12) but not BODIPY-FLC(5) to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC(12) to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC(12) was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Processing and characterization of device solder interconnection and module attachment for power electronics modules

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Haque, Shatil

This research is focused on the processing of an innovative three-dimensional packaging architecture for power electronics building blocks with soldered device interconnections and subsequent characterization of the module's critical interfaces. A low-cost approach termed metal posts interconnected parallel plate structure (MPIPPS) was developed for packaging high-performance modules of power electronics building blocks (PEBB). The new concept implemented direct bonding of copper posts, not wire bonding of fine aluminum wires, to interconnect power devices as well as joining the different circuit planes together. We have demonstrated the feasibility of this packaging approach by constructing PEBB modules (consisting of Insulated Gate Bipolar Transistors (IGBTs), diodes, and a few gate driver elements and passive components). In the 1st phase of module fabrication with IGBTs with Si3N 4 passivation, we had successfully fabricated packaged devices and modules using the MPIPPS technique. These modules were tested electrically and thermally, and they operated at pulse-switch and high power stages up to 6kW. However, in the 2nd phase of module fabrication with polyimide passivated devices, we experienced significant yield problems due to metallization difficulties of these devices. The under-bump metallurgy scheme for the development of a solderable interface involved sputtering of Ti-Ni-Cu and Cr-Cu, and an electroless deposition of Zn-Ni-Au metallization. The metallization process produced excellent yield in the case of Si3N4 passivated devices. However, under the same metallization schemes, devices with a polyimide passivation exhibited inconsistent electrical contact resistance. We found that organic contaminants such as hydrocarbons remain in the form of thin monolayers on the surface, even in the case of as-received devices from the manufacturer. Moreover, in the case of polyimide passivated devices, plasma cleaning introduced a few carbon constituents on the

TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements.

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Walsh, Mary F; Ampasala, Dinakar R; Rishi, Arun K; Basson, Marc D

2009-02-01

TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.

Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

Science.gov (United States)

Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

2016-01-01

Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

Genome-wide identification of basic helix-loop-helix and NF-1 motifs underlying GR binding sites in male rat hippocampus

DEFF Research Database (Denmark)

Pooley, John R.; Flynn, Ben P.; Grøntved, Lars

2017-01-01

linked to structural and organizational roles, an absence of major tethering partners for GRs, and little or no evidence for binding at negative glucocorticoid response elements. A basic helix-loop-helix motif closely resembling a NeuroD1 or Olig2 binding site was found underlying a subset of GR binding......Glucocorticoids regulate hippocampal function in part by modulating gene expression through the glucocorticoid receptor (GR). GR binding is highly cell type specific, directed to accessible chromatin regions established during tissue differentiation. Distinct classes of GR binding sites...

Cell Surface Binding and Internalization of Aβ Modulated by Degree of Aggregation

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David A. Bateman

2011-01-01

Full Text Available The amyloid peptides, Aβ40 and Aβ42, are generated through endoproteolytic cleavage of the amyloid precursor protein. Here we have developed a model to investigate the interaction of living cells with various forms of aggregated Aβ40/42. After incubation at endosomal pH 6, we observed a variety of Aβ conformations after 3 (Aβ3, 24 (Aβ24, and 90 hours (Aβ90. Both Aβ4224 and Aβ4024 were observed to rapidly bind and internalize into differentiated PC12 cells, leading to accumulation in the lysosome. In contrast, Aβ40/4290 were both found to only weakly associate with cells, but were observed as the most aggregated using dynamic light scattering and thioflavin-T. Internalization of Aβ40/4224 was inhibited with treatment of monodansylcadaverine, an endocytosis inhibitor. These studies indicate that the ability of Aβ40/42 to bind and internalize into living cells increases with degree of aggregation until it reaches a maximum beyond which its ability to interact with cells diminishes drastically.

Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

Science.gov (United States)

Stax, Martijn J; Mouser, Emily E I M; van Montfort, Thijs; Sanders, Rogier W; de Vries, Henry J C; Dekker, Henk L; Herrera, Carolina; Speijer, Dave; Pollakis, Georgios; Paxton, William A

2015-01-01

Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

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Christopher J Brown

Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

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Jean-Marc Taymans

Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4β2 nicotinic acetylcholine receptors

DEFF Research Database (Denmark)

Olsen, Jeppe A; Ahring, Philip K; Kastrup, Jette Sandholm Jensen

2014-01-01

Modulation of Cys loop receptor ion channels is a proven drug discovery strategy, but many underlying mechanisms of the mode of action are poorly understood. We report the x-ray structure of the acetylcholine-binding protein from Lymnaea stagnalis with NS9283, a stoichiometry selective positive...... on efficacy. The shared modulatory profile along with a binding site located in an extracellular subunit interface suggest that modulation via an agonist-like mechanism may be a common mechanism of action that potentially could apply to Cys loop receptors beyond the α4β2 nAChRs....... modulator that targets the α4-α4 interface of α4β2 nicotinic acetylcholine receptors (nAChRs). Together with homology modeling, mutational data, quantum mechanical calculations, and pharmacological studies on α4β2 nAChRs, the structure reveals a modulator binding mode that overlaps the α4-α4 interface...

The Extracellular Protein Factor Epf from Streptococcus pyogenes Is a Cell Surface Adhesin That Binds to Cells through an N-terminal Domain Containing a Carbohydrate-binding Module*

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Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H. P.; Whisstock, James C.; Baker, Edward N.; Kreikemeyer, Bernd

2012-01-01

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain. PMID:22977243

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

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Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

2016-07-01

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei[S

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Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J.

2016-01-01

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC12 but not BODIPY-FLC5 to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC12 to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC12 was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. PMID:26658423

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

DEFF Research Database (Denmark)

Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

2006-01-01

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

A peptide derived from a trans-homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

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Køhler, Lene B; Soroka, Vladislav; Korshunova, Irina

2010-01-01

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, t...

How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

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Dudev, Todor; Grauffel, Cédric; Lim, Carmay

2017-02-01

Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

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Tsai, Kuang-Lei; Sato, Shigeo; Tomomori-Sato, Chieri; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2013-01-01

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism. PMID:23563140

De-escalatory confidence-building measures after START

International Nuclear Information System (INIS)

Stockton, P.N.

1992-01-01

The very notion of examining de-escalatory CBMs in the context of the START negotiations appears odd. Given the immense progress in US-Soviet relations, of which the proposed START agreement is emblematic, the risk of the two nations stumbling to the brink of war seems increasingly remote. Why bother analyzing CBMs designed to help resolve such an improbable event? The obvious answer is that relations between the US and the Soviet Union - or whatever array of nations succeed it - could take an unforeseeable turn for the worse. Once ratified, the START agreement is supposed to remain in force for seven years, more than enough time for political tensions to return with a vengeance. This suggests a more prudent question: if our two nations do stumble to the brink of war, will the START agreement affect the prospect of de-escalatory CBMs? And if the US and Soviet Union pursue deeper force-reductions in a follow-on START II agreement, could that agreement be designed to facilitate more effective de-escalatory measures? This paper sets the stage for addressing such questions by outlining the START agreement and offering a brief overview of how START might affect the prospects for CBMs. Then, after examining some specific CBM proposals in light of START, an analysis will be made of the de-escalatory problems and opportunities that a Start II treaty might create

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

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Hong, Weimin Conrad; Yano, Hideaki; Hiranita, Takato; Chin, Frederick T; McCurdy, Christopher R; Su, Tsung-Ping; Amara, Susan G; Katz, Jonathan L

2017-07-07

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ 1 R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ 1 R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di- o -tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the B max values of [ 3 H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ 1 R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ 1 R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ 1 R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ 1 R antagonist CM304. Moreover, σ 1 R ligands had distinct effects on σ 1 R multimerization. CM304 increased the proportion of multimeric σ 1 Rs, whereas (+)-pentazocine increased monomeric σ 1 Rs. Together these results support the hypothesis that σ 1 R agonists promote dissociation of σ 1 R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ 1 R agonists in animal models. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Laminin-binding integrins and their tetraspanin partners as potential antimetastatic targets

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Stipp, Christopher S.

2010-01-01

Within the integrin family of cell adhesion receptors, integrins α3β1, α6β1, α6β4 and α7β1 make up a laminin-binding subfamily. The literature is divided on the role of these laminin-binding integrins in metastasis, with different studies indicating either pro- or antimetastatic functions. The opposing roles of the laminin-binding integrins in different settings might derive in part from their unusually robust associations with tetraspanin proteins. Tetraspanins organise integrins into multiprotein complexes within discrete plasma membrane domains termed tetraspanin-enriched microdomains (TEMs). TEM association is crucial to the strikingly rapid cell migration mediated by some of the laminin-binding integrins. However, emerging data suggest that laminin-binding integrins also promote the stability of E-cadherin-based cell–cell junctions, and that tetraspanins are essential for this function as well. Thus, TEM association endows the laminin-binding integrins with both pro-invasive functions (rapid migration) and anti-invasive functions (stable cell junctions), and the composition of TEMs in different cell types might help determine the balance between these opposing activities. Unravelling the tetraspanin control mechanisms that regulate laminin-binding integrins will help to define the settings where inhibiting the function of these integrins would be helpful rather than harmful, and may create opportunities to modulate integrin activity in more sophisticated ways than simple functional blockade. PMID:20078909

Low 5-HT1B receptor binding in the migraine brain

DEFF Research Database (Denmark)

Deen, Marie; Hansen, Hanne D; Hougaard, Anders

2018-01-01

Background The pathophysiology of migraine may involve dysfunction of serotonergic signaling. In particular, the 5-HT1B receptor is considered a key player due to the efficacy of 5-HT1B receptor agonists for treatment of migraine attacks. Aim To examine the cerebral 5-HT1B receptor binding....... Patients who reported migraine brain regions involved in pain modulation as regions of interest and applied a latent variable model (LVM) to assess the group effect on binding across these regions. Results Our data...... support a model wherein group status predicts the latent variable ( p = 0.038), with migraine patients having lower 5-HT1B receptor binding across regions compared to controls. Further, in a whole-brain voxel-based analysis, time since last migraine attack correlated positively with 5-HT1B receptor...

PROCARB: A Database of Known and Modelled Carbohydrate-Binding Protein Structures with Sequence-Based Prediction Tools

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Adeel Malik

2010-01-01

Full Text Available Understanding of the three-dimensional structures of proteins that interact with carbohydrates covalently (glycoproteins as well as noncovalently (protein-carbohydrate complexes is essential to many biological processes and plays a significant role in normal and disease-associated functions. It is important to have a central repository of knowledge available about these protein-carbohydrate complexes as well as preprocessed data of predicted structures. This can be significantly enhanced by tools de novo which can predict carbohydrate-binding sites for proteins in the absence of structure of experimentally known binding site. PROCARB is an open-access database comprising three independently working components, namely, (i Core PROCARB module, consisting of three-dimensional structures of protein-carbohydrate complexes taken from Protein Data Bank (PDB, (ii Homology Models module, consisting of manually developed three-dimensional models of N-linked and O-linked glycoproteins of unknown three-dimensional structure, and (iii CBS-Pred prediction module, consisting of web servers to predict carbohydrate-binding sites using single sequence or server-generated PSSM. Several precomputed structural and functional properties of complexes are also included in the database for quick analysis. In particular, information about function, secondary structure, solvent accessibility, hydrogen bonds and literature reference, and so forth, is included. In addition, each protein in the database is mapped to Uniprot, Pfam, PDB, and so forth.

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

Science.gov (United States)

Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

2016-04-01

Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

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Gunawardane, Ruwanthi N.; Fordstrom, Preston; Piper, Derek E.; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

2016-01-01

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

Exploring the impact of the side-chain length on peptide/RNA binding events.

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Sbicca, Lola; González, Alejandro López; Gresika, Alexandra; Di Giorgio, Audrey; Closa, Jordi Teixido; Tejedor, Roger Estrada; Andréola, Marie-Line; Azoulay, Stéphane; Patino, Nadia

2017-07-19

The impact of the amino-acid side-chain length on peptide-RNA binding events has been investigated using HIV-1 Tat derived peptides as ligands and the HIV-1 TAR RNA element as an RNA model. Our studies demonstrate that increasing the length of all peptide side-chains improves unexpectedly the binding affinity (K D ) but reduces the degree of compactness of the peptide-RNA complex. Overall, the side-chain length appears to modulate in an unpredictable way the ability of the peptide to compete with the cognate TAR RNA partner. Beyond the establishment of non-intuitive fundamental relationships, our results open up new perspectives in the design of effective RNA ligand competitors, since a large number of them have already been identified but few studies report on the modulation of the biological activity by modifying in the same way the length of all chains connecting RNA recognition motives to the central scaffold of a ligand.

Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17β-estradiol-induced transcriptional activity.

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Pesiri, Valeria; La Rosa, Piergiorgio; Stano, Pasquale; Acconcia, Filippo

2013-06-15

Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.

Prioritization of gene regulatory interactions from large-scale modules in yeast

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Bringas Ricardo

2008-01-01

Full Text Available Abstract Background The identification of groups of co-regulated genes and their transcription factors, called transcriptional modules, has been a focus of many studies about biological systems. While methods have been developed to derive numerous modules from genome-wide data, individual links between regulatory proteins and target genes still need experimental verification. In this work, we aim to prioritize regulator-target links within transcriptional modules based on three types of large-scale data sources. Results Starting with putative transcriptional modules from ChIP-chip data, we first derive modules in which target genes show both expression and function coherence. The most reliable regulatory links between transcription factors and target genes are established by identifying intersection of target genes in coherent modules for each enriched functional category. Using a combination of genome-wide yeast data in normal growth conditions and two different reference datasets, we show that our method predicts regulatory interactions with significantly higher predictive power than ChIP-chip binding data alone. A comparison with results from other studies highlights that our approach provides a reliable and complementary set of regulatory interactions. Based on our results, we can also identify functionally interacting target genes, for instance, a group of co-regulated proteins related to cell wall synthesis. Furthermore, we report novel conserved binding sites of a glycoprotein-encoding gene, CIS3, regulated by Swi6-Swi4 and Ndd1-Fkh2-Mcm1 complexes. Conclusion We provide a simple method to prioritize individual TF-gene interactions from large-scale transcriptional modules. In comparison with other published works, we predict a complementary set of regulatory interactions which yields a similar or higher prediction accuracy at the expense of sensitivity. Therefore, our method can serve as an alternative approach to prioritization for

Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

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Ren, Weitong; Ji, Dongqing; Xu, Xiulian

2018-01-01

The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

Directory of Open Access Journals (Sweden)

Weitong Ren

Full Text Available The HIV-1 nucleocapsid 7 (NCp7 plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3 recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells

DEFF Research Database (Denmark)

Ma, Ying Jie; Kang, Hee Jung; Kim, Ji Yeon

2013-01-01

It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...... cytokine production by LPS-mediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells.......It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...

Solution structure of the twelfth cysteine-rich ligand-binding repeat in rat megalin

International Nuclear Information System (INIS)

Wolf, Christian A.; Dancea, Felician; Shi Meichen; Bade-Noskova, Veronika; Rueterjans, Heinz; Kerjaschki, Dontscho; Luecke, Christian

2007-01-01

Megalin, an approx. 600 kDa transmembrane glycoprotein that acts as multi-ligand transporter, is a member of the low density lipoprotein receptor gene family. Several cysteine-rich repeats, each consisting of about 40 residues, are responsible for the multispecific binding of ligands. The solution structure of the twelfth cysteine-rich ligand-binding repeat with class A motif found in megalin features two short β-strands and two helical turns, yielding the typical fold with a I-III, II-V and IV-VI disulfide bridge connectivity pattern and a calcium coordination site at the C-terminal end. The resulting differences in electrostatic surface potential compared to other ligand-binding modules of this gene family, however, may be responsible for the functional divergence

Comparison of crystal and solution hemoglobin binding of selected antigelling agents and allosteric modifiers

International Nuclear Information System (INIS)

Mehanna, A.S.; Abraham, D.J.

1990-01-01

This paper details comprehensive binding studies (solution and X-ray) of human hemoglobin A with a group of halogenated carboxylic acids that were investigated as potential antisickling agents. It is, to our knowledge, the first study to compare solution and crystal binding for a series of compounds under similar high-salt conditions used for cocrystallization. The compounds include [(3,4-dichlorobenzyl)oxy]acetic acid, [(p-bromobenzyl)oxy]acetic acid, clofibric acid, and bezafibrate. The location and stereochemistry of binding sites have been established by X-ray crystallography, while the number of binding sites and affinity constants were measured by using equilibrium dialysis. The observed crystal structures are consistent with the binding observed in solution and that the number of binding sites is independent of salt concentration, while the binding constant increases with increasing salt concentration. The studies also reveal that relatively small changes in the chemical structure of a drug molecule can result in entirely different binding sites on the protein. Moreover, the X-ray studies provide a possible explanation for the multiplicity in function exhibited by these compounds as allosteric modulators and/or antisickling agents. Finally, the studies indicate that these compounds bind differently to the R and T states of hemoglobin, and observation of special significance to the original design of these agents

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin*♦

Science.gov (United States)

Bensing, Barbara A.; Loukachevitch, Lioudmila V.; McCulloch, Kathryn M.; Yu, Hai; Vann, Kendra R.; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M.; Iverson, T. M.

2016-01-01

Streptococcus sanguinis is a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets. S. sanguinis expresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. PMID:26833566

Coco is a dual activity modulator of TGFβ signaling

Science.gov (United States)

Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

2015-01-01

The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

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Felipe Merino

2015-06-01

Full Text Available Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

Science.gov (United States)

Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

2014-02-10

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties. Copyright © 2013 Elsevier B.V. All rights reserved.

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins.

Science.gov (United States)

Raman, Rajeev; Rajanikanth, V; Palaniappan, Raghavan U M; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P; Sharma, Yogendra; Chang, Yung-Fu

2010-12-29

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th) (Lig A9) and 10(th) repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

PICALM modulates autophagy activity and tau accumulation

Science.gov (United States)

Moreau, Kevin; Fleming, Angeleen; Imarisio, Sara; Lopez Ramirez, Ana; Mercer, Jacob L.; Jimenez-Sanchez, Maria; Bento, Carla F.; Puri, Claudia; Zavodszky, Eszter; Siddiqi, Farah; Lavau, Catherine P.; Betton, Maureen; O’Kane, Cahir J.; Wechsler, Daniel S.; Rubinsztein, David C.

2014-01-01

Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover. PMID:25241929

A Common Structural Component for β-Subunit Mediated Modulation of Slow Inactivation in Different KV Channels

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Nathalie Strutz-Seebohm

2013-06-01

Full Text Available Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

RNA binding efficacy of theophylline, theobromine and caffeine.

Science.gov (United States)

Johnson, I Maria; Kumar, S G Bhuvan; Malathi, R

2003-04-01

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.

Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

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Martijn J Stax

Full Text Available Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa, heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments and its receptor (intelectin-1 as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice

International Nuclear Information System (INIS)

Watanabe, Kenta; Hirata, Michiko; Tominari, Tsukasa; Matsumoto, Chiho; Endo, Yasuyuki; Murphy, Gillian; Nagase, Hideaki

2016-01-01

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERβ. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male. - Highlights: • A novel carborane compound BA321 binds to both AR and ERs, ERα and ERβ. • BA321 restores bone loss in orchidectomized mice without effects on sex organ. • BA321 acts as an estrogen agonist in bone and uterus in ovariectomized mice. • BA321 may be a new SARM to prevent the loss of musculoskeletal mass in elder men.

BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice

Energy Technology Data Exchange (ETDEWEB)

Watanabe, Kenta [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Hirata, Michiko [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Tominari, Tsukasa [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Matsumoto, Chiho [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Endo, Yasuyuki [Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, 981-8558 (Japan); Murphy, Gillian [Department of Oncology, University of Cambridge, Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge, CB2 0RE (United Kingdom); Nagase, Hideaki [Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 (Japan); Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, OX3 7FY (United Kingdom); and others

2016-09-09

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERβ. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male. - Highlights: • A novel carborane compound BA321 binds to both AR and ERs, ERα and ERβ. • BA321 restores bone loss in orchidectomized mice without effects on sex organ. • BA321 acts as an estrogen agonist in bone and uterus in ovariectomized mice. • BA321 may be a new SARM to prevent the loss of musculoskeletal mass in elder men.

Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

International Nuclear Information System (INIS)

Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

2010-01-01

Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

Insulin stimulation of [3H]-ouabain binding to cerebrovascular (Na+ + K+)-ATPase

International Nuclear Information System (INIS)

Caspers, M.L.; Grammas, P.

1986-01-01

Brain microvessels were isolated from rat cerebral cortices. The binding of [ 3 H]-ouabain to microvascular (Na + + K + )-ATPase increased with microvessel protein (37-110μg) and was time dependent with maximum binding observed at 15 min at 37 0 C. Non-specific binding, measured in the presence of 50μM ouabain, was less than 2% of total binding. Scatchard analysis of preliminary [ 3 H]-ouabain binding data yielded a K/sub D/ of 44nM and a B/sub max/ of 12pmol/mg. Since the high affinity (α+) form of the enzyme is purportedly hormonally regulated, the effect of insulin on [ 3 H]-ouabain binding to microvessels was studied. Insulin (0.001-10μM) stimulation of [ 3 H]-ouabain binding was dose dependent. To assess whether this was a specific or a peptide-protective effect, assays were performed in the presence of bovine serum albumin (BSA). Addition of BSA (10μM) enhanced the amount of [ 3 H]-ouabain bound 4-fold. Further increases in the BSA concentration (20μM) did not increase binding. Addition of 10μM insulin evoked a 20% increase in [ 3 H]-ouabain binding above BSA-treated controls. In summary, the data suggest that the (α+) form of the (Na + + K + )-ATPase is present in cerebral endothelium and [ 3 H]-ouabain binding is significantly elevated by insulin in a dose dependent manner. Therefore, insulin may regulate microvascular (Na + + K + )-ATPase and thus be a modulator of blood-brain permeability to ions

Finding cis-regulatory modules in Drosophila using phylogenetic hidden Markov models

DEFF Research Database (Denmark)

Wong, Wendy S W; Nielsen, Rasmus

2007-01-01

MOTIVATION: Finding the regulatory modules for transcription factors binding is an important step in elucidating the complex molecular mechanisms underlying regulation of gene expression. There are numerous methods available for solving this problem, however, very few of them take advantage of th...

The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

Science.gov (United States)

Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

2007-09-01

Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

Structural determinants for binding to angiotensin converting enzyme 2 (ACE2 and angiotensin receptors

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Daniel eClayton

2015-01-01

Full Text Available Angiotensin converting enzyme 2 (ACE2 is a zinc carboxypeptidase involved in the renin angiotensin system (RAS and inactivates the potent vasopressive peptide angiotensin II (Ang II by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R and angiotensin type 2 (AT2R receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here we further explore this region for the potential to modulate ligand specificity. In this study, 1 a library of forty-seven peptides derived from the C-terminal tetra-peptide sequence (-IHPF of Ang II was synthesised and assessed for ACE2 binding, 2 the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, 3 high affinity ACE2 binding chimeric AngII analogues were then synthesized and assessed, 4 the structure of the full-length Ang II analogues were assessed by circular dichroism, and 5 the Ang II analogues were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor

DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

Science.gov (United States)

Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

2010-06-18

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Structure of Drosophila Oskar reveals a novel RNA binding protein

Science.gov (United States)

Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

2015-01-01

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function

International Nuclear Information System (INIS)

Marshman, Emma; Streuli, Charles H

2002-01-01

Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development in the mammary gland during puberty and pregnancy. IGFs interact with IGF-binding proteins and regulate their function. The present review focuses on the role of IGFs and IGF-binding proteins in the mammary gland and describes how modulation of their actions occurs by association with hormones, other growth factors and the extracellular matrix. The review will also highlight the involvement of the IGF axis in breast cancer

Lysozyme binding ability toward psychoactive stimulant drugs: Modulatory effect of colloidal metal nanoparticles.

Science.gov (United States)

Sonu, Vikash K; Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Mitra, Sivaprasad

2016-10-01

The interaction and binding behavior of the well-known psychoactive stimulant drugs theophylline (THP) and theobromine (THB) with lysozyme (LYS) was monitored by in-vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of the drugs is due to the formation of protein-drug complex in the ground state in both the cases. However, the binding interaction is almost three orders of magnitude stronger in THP, which involves mostly hydrogen bonding interaction in comparison with THB where hydrophobic binding plays the predominant role. The mechanism of fluorescence quenching (static type) remains same also in presence of gold and silver nanoparticles (NPs); however, the binding capacity of LYS with the drugs changes drastically in comparison with that in aqueous buffer medium. While the binding affinity of LYS to THB increases ca. 100 times in presence of both the NPs, it is seen to decrease drastically (by almost 1000 fold) for THP. This significant modulation in binding behavior indicates that the drug transportation capacity of LYS can be controlled significantly with the formation protein-NP noncovalent assembly system as an efficient delivery channel. Copyright © 2016 Elsevier B.V. All rights reserved.

Divers models of divalent cation interaction to calcium-binding proteins: techniques and anthology.

Science.gov (United States)

Cox, Jos A

2013-01-01

Intracellular Ca(2+)-binding proteins (CaBPs) are sensors of the calcium signal and several of them even shape the signal. Most of them are equipped with at least two EF-hand motifs designed to bind Ca(2+). Their affinities are very variable, can display cooperative effects, and can be modulated by physiological Mg(2+) concentrations. These binding phenomena are monitored by four major techniques: equilibrium dialysis, fluorimetry with fluorescent Ca(2+) indicators, flow dialysis, and isothermal titration calorimetry. In the last quarter of the twentieth century reports on the ion-binding characteristics of several abundant wild-type CaBPs were published. With the advent of recombinant CaBPs it became possible to determine these properties on previously inaccessible proteins. Here I report on studies by our group carried out in the last decade on eight families of recombinant CaBPs, their mutants, or truncated domains. Moreover this chapter deals with the currently used methods for quantifying the binding of Ca(2+) and Mg(2+) to CaBPs.

The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials.

Science.gov (United States)

Sato, Kanna; Ito, Sachiko; Fujii, Takeo; Suzuki, Ryu; Takenouchi, Sachi; Nakaba, Satoshi; Funada, Ryo; Sano, Yuzou; Kajita, Shinya; Kitano, Hidemi; Katayama, Yoshihiro

2010-11-01

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.

Functional evolution of cis-regulatory modules at a homeotic gene in Drosophila.

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Margaret C W Ho

2009-11-01

Full Text Available It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs. How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets

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Singh, Anirudh K.; Woodiga, Shireen A.; Grau, Margaret A.

2016-01-01

ABSTRACT Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. PMID:27993975

Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome.

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Dresch, Jacqueline M; Zellers, Rowan G; Bork, Daniel K; Drewell, Robert A

2016-01-01

A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.

Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

International Nuclear Information System (INIS)

Gurwitz, D.; Egozi, Y.; Henis, Y.I.; Kloog, Y.; Sokolovsky, M.

1987-01-01

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [ 3 H]-antagonist as well as of the labeled natural neurotransmitter, [ 3 H]-acetylcholine [( 3 H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [ 3 H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [ 3 H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [ 3 H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [ 3 H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms

NCS-1 associates with adenosine A2A receptors and modulates receptor function

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Gemma eNavarro

2012-04-01

Full Text Available Modulation of G protein-coupled receptor (GPCR signalling by local changes in intracellular calcium concentration is an established function of Calmodulin which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with calmodulin targets with different functional outcome. In the present study we aimed to investigate if a target of calmodulin – the A2A adenosine receptor, is able to associate with two other neuronal calcium binding proteins, namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer and co-immunoprecipitation experiments we show the existence of A2A - NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signalling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signalling.

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig proteins.

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Rajeev Raman

Full Text Available BACKGROUND: Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. PRINCIPAL FINDINGS: We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th (Lig A9 and 10(th repeats (Lig A10; and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon. All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm, probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. CONCLUSIONS: We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

Binding of MCM-interacting proteins to ATP-binding site in MCM6

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Hosoi A

2016-03-01

Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

Cholesterol Oxidase Binds TLR2 and Modulates Functional Responses of Human Macrophages

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Katarzyna Bednarska

2014-01-01

Full Text Available Cholesterol oxidase (ChoD is considered to be an important virulence factor for Mycobacterium tuberculosis (Mtb, but its influence on macrophage activity is unknown. Here we used Nocardia erythropolis ChoD, which is very similar to the Mtb enzyme (70% identity at the amino-acid level, to evaluate the impact of bacterial ChoD on the activity of THP-1-derived macrophages in vitro. We found that ChoD decreased the surface expression of Toll-like receptor type 2 (TLR2 and complement receptor 3 (CR3 on these macrophages. Flow cytometry and confocal microscopy showed that ChoD competed with lipoteichoic acid for ligand binding sites on TLR2 but not on CR3, suggesting that ChoD signaling is mediated via TLR2. Binding of ChoD to the membrane of macrophages had diverse effects on the activity of macrophages, activating p38 mitogen activated kinase and stimulating production of a large amount of interleukin-10. Moreover, ChoD primed macrophages to enhance the production of reactive oxygen species in response to the phorbol myristate acetate, which was reduced by “switching off” TLR-derived signaling through interleukin-1 receptor-associated kinases 1 and 4 inhibition. Our study revealed that ChoD interacts directly with macrophages via TLR2 and influences the biological activity of macrophages during the development of the initial response to infection.

Mapping small molecule binding data to structural domains.

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Kruger, Felix A; Rostom, Raghd; Overington, John P

2012-01-01

Large-scale bioactivity/SAR Open Data has recently become available, and this has allowed new analyses and approaches to be developed to help address the productivity and translational gaps of current drug discovery. One of the current limitations of these data is the relative sparsity of reported interactions per protein target, and complexities in establishing clear relationships between bioactivity and targets using bioinformatics tools. We detail in this paper the indexing of targets by the structural domains that bind (or are likely to bind) the ligand within a full-length protein. Specifically, we present a simple heuristic to map small molecule binding to Pfam domains. This profiling can be applied to all proteins within a genome to give some indications of the potential pharmacological modulation and regulation of all proteins. In this implementation of our heuristic, ligand binding to protein targets from the ChEMBL database was mapped to structural domains as defined by profiles contained within the Pfam-A database. Our mapping suggests that the majority of assay targets within the current version of the ChEMBL database bind ligands through a small number of highly prevalent domains, and conversely the majority of Pfam domains sampled by our data play no currently established role in ligand binding. Validation studies, carried out firstly against Uniprot entries with expert binding-site annotation and secondly against entries in the wwPDB repository of crystallographic protein structures, demonstrate that our simple heuristic maps ligand binding to the correct domain in about 90 percent of all assessed cases. Using the mappings obtained with our heuristic, we have assembled ligand sets associated with each Pfam domain. Small molecule binding has been mapped to Pfam-A domains of protein targets in the ChEMBL bioactivity database. The result of this mapping is an enriched annotation of small molecule bioactivity data and a grouping of activity classes

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

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Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

In search of allosteric modulators of a7-nAChR by solvent density guided virtual screening.

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Dey, Raja; Chen, Lin

2011-04-01

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels whose activity can be modulated by endogenous neurotransmitters as well as by synthetic ligands that bind the same or distinct sites from the natural ligand. The subtype of α7 nAChR has been considered as a potenial therapeutic target for Alzheimer's disease, schizophrenia and other neurological and psychiatric disorders. Here we have developed a homology model of α7 nAChR based on two high resolution crystal structures with Brookhaven Protein Data Bank (PDB) codes 2QC1 and 2WN9 for threading on one monomer and then for building a pentamer, respectively. A number of small molecule binding sites are identified using Pocket Finder (J. An, M. Tortov, and R. Abagyan, Molecular & Cellular Proteomics, 4.6, 752-761 (2005)) of Internal Coordinate Mechanics (ICM). Remarkably, these computer-identified sites match perfectly with ordered solvent densities found in the high-resolution crystal structure of α1 nAChR, suggesting that the surface cavities in the α7 nAChR model are likely binding sites of small molecules. A high throughput virtual screening by flexible ligand docking of 5008 small molecule compounds was performed at three potential allosteric modulator (AM) binding sites of α7 nAChR using Molsoft ICM software (R. Abagyan, M. Tortov and D. Kuznetsov, J Comput Chem 15, 488-506, (1994)). Some experimentally verified allosteric modulators of α7 like CCMI comp-6, LY 7082101, 5-HI, TQS, PNU-120596, genistein, and NS-1738 ranked among top 100 compounds, while the rest of the compounds in the list could guide further search for new allosteric modulators.

New insights into structure and function of the different types of fatty acid-binding protein

NARCIS (Netherlands)

Zimmerman, Augusta Wilhelmina

2002-01-01

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. They may also modulate the effect of fatty acids on various metabolic enzymes and receptors and on cellular

Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

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Miroslav M. Savic

2005-01-01

Full Text Available Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α5 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states.

Structural determination of functional units of the nucleotide binding domain (NBD94 of the reticulocyte binding protein Py235 of Plasmodium yoelii.

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Ardina Grüber

2010-02-01

Full Text Available Invasion of the red blood cells (RBC by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94 of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547, NBD94(566-663 and NBD94(674-793, respectively. Using fluorescence correlation spectroscopy NBD94(444-547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663 and NBD94(674-793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663 consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793 in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the

Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

Energy Technology Data Exchange (ETDEWEB)

Baltazar, Ludmila M.; Nakayasu, Ernesto S.; Sobreira, Tiago; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.

2016-03-30

Histoplasma capsulatumproduces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment ofH. capsulatumcells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bindH. capsulatumheat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion.

IMPORTANCEDiverse fungal species release extracellular vesicles, indicating that this is a

Interaction of Object Binding Cues in Binaural Masking Pattern Experiments.

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Verhey, Jesko L; Lübken, Björn; van de Par, Steven

2016-01-01

Object binding cues such as binaural and across-frequency modulation cues are likely to be used by the auditory system to separate sounds from different sources in complex auditory scenes. The present study investigates the interaction of these cues in a binaural masking pattern paradigm where a sinusoidal target is masked by a narrowband noise. It was hypothesised that beating between signal and masker may contribute to signal detection when signal and masker do not spectrally overlap but that this cue could not be used in combination with interaural cues. To test this hypothesis an additional sinusoidal interferer was added to the noise masker with a lower frequency than the noise whereas the target had a higher frequency than the noise. Thresholds increase when the interferer is added. This effect is largest when the spectral interferer-masker and masker-target distances are equal. The result supports the hypothesis that modulation cues contribute to signal detection in the classical masking paradigm and that these are analysed with modulation bandpass filters. A monaural model including an across-frequency modulation process is presented that account for this effect. Interestingly, the interferer also affects dichotic thresholds indicating that modulation cues also play a role in binaural processing.

India-Pakistan: nuclear stability and diplomacy

International Nuclear Information System (INIS)

Mishra, Rajesh Kumar

2005-01-01

The conceptual discourse, contributed to in the main by Western scholars, on the security and strategic stability of new nuclear weapon states like India and Pakistan seems alarmist. In reality, however, India and Pakistan have been mutually deliberating on various aspects of nuclear confidence-building measures (CBMs). This article is an effort to identify the issues of nuclear security concerns in two spheres - academic and policy formulations. The emphasis is more on the nuclear thinking of the two countries and the diplomatic challenges ahead particularly on the nuclear CBMs. (author)

Peroxisome Proliferators-Activated Receptor (PPAR Modulators and Metabolic Disorders

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Min-Chul Cho

2008-01-01

Full Text Available Overweight and obesity lead to an increased risk for metabolic disorders such as impaired glucose regulation/insulin resistance, dyslipidemia, and hypertension. Several molecular drug targets with potential to prevent or treat metabolic disorders have been revealed. Interestingly, the activation of peroxisome proliferator-activated receptor (PPAR, which belongs to the nuclear receptor superfamily, has many beneficial clinical effects. PPAR directly modulates gene expression by binding to a specific ligand. All PPAR subtypes (α,γ, and σ are involved in glucose metabolism, lipid metabolism, and energy balance. PPAR agonists play an important role in therapeutic aspects of metabolic disorders. However, undesired effects of the existing PPAR agonists have been reported. A great deal of recent research has focused on the discovery of new PPAR modulators with more beneficial effects and more safety without producing undesired side effects. Herein, we briefly review the roles of PPAR in metabolic disorders, the effects of PPAR modulators in metabolic disorders, and the technologies with which to discover new PPAR modulators.

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

International Nuclear Information System (INIS)

Ritvos, O.; Ranta, T.; Jalkanen, J.; Suikkari, A.M.; Voutilainen, R.; Bohn, H.; Rutanen, E.M.

1988-01-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells

Confidence building measures at sea:opportunities for India and Pakistan.

Energy Technology Data Exchange (ETDEWEB)

Vohra, Ravi Bhushan Rear Admiral (; ); Ansari, Hasan Masood Rear Admiral (; )

2003-12-01

The sea presents unique possibilities for implementing confidence building measures (CBMs) between India and Pakistan that are currently not available along the contentious land borders surrounding Jammu and Kashmir. This is due to the nature of maritime issues, the common military culture of naval forces, and a less contentious history of maritime interaction between the two nations. Maritime issues of mutual concern provide a strong foundation for more far-reaching future CBMs on land, while addressing pressing security, economic, and humanitarian needs at sea in the near-term. Although Indian and Pakistani maritime forces currently have stronger opportunities to cooperate with one another than their counterparts on land, reliable mechanisms to alleviate tension or promote operational coordination remain non-existent. Therefore, possible maritime CBMs, as well as pragmatic mechanisms to initiate and sustain cooperation, require serious examination. This report reflects the unique joint research undertaking of two retired Senior Naval Officers from both India and Pakistan, sponsored by the Cooperative Monitoring Center of the International Security Center at Sandia National Laboratories. Research focuses on technology as a valuable tool to facilitate confidence building between states having a low level of initial trust. Technical CBMs not only increase transparency, but also provide standardized, scientific means of interacting on politically difficult problems. Admirals Vohra and Ansari introduce technology as a mechanism to facilitate consistent forms of cooperation and initiate discussion in the maritime realm. They present technical CBMs capable of being acted upon as well as high-level political recommendations regarding the following issues: (1) Delimitation of the maritime boundary between India and Pakistan and its relationship to the Sir Creek dispute; (2) Restoration of full shipping links and the security of ports and cargos; (3) Fishing within

Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action

Energy Technology Data Exchange (ETDEWEB)

Gee, K.W.; Bolger, M.B.; Brinton, R.E.; Coirini, H.; McEwen, B.S.

1988-08-01

Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by (35S)-t-butylbicyclophosphorothionate ((35S)TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of (35S)TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of (35S)TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates (35S)TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All (35S)TBPS binding sites appear to be functionally coupled to a steroid modulatory site. Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of (3H)promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.

Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

International Nuclear Information System (INIS)

Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J.

1990-01-01

We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed

Small Molecule Microarrays Enable the Identification of a Selective, Quadruplex-Binding Inhibitor of MYC Expression.

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Felsenstein, Kenneth M; Saunders, Lindsey B; Simmons, John K; Leon, Elena; Calabrese, David R; Zhang, Shuling; Michalowski, Aleksandra; Gareiss, Peter; Mock, Beverly A; Schneekloth, John S

2016-01-15

The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small

The Ala54Thr Polymorphism of the Fatty Acid Binding Protein 2 Gene Modulates HDL Cholesterol in Mexican-Americans with Type 2 Diabetes

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Lorena M. Salto

2015-12-01

Full Text Available The alanine to threonine amino acid substitution at codon 54 (Ala54Thr of the intestinal fatty acid binding protein (FABP2 has been associated with elevated levels of insulin and blood glucose as well as with dyslipidemia. The aim of this study was to characterize the effect of this FABP2 polymorphism in Mexican-Americans with type 2 diabetes (T2D in the context of a three-month intervention to determine if the polymorphism differentially modulates selected clinical outcomes. For this study, we genotyped 43 participant samples and performed post-hoc outcome analysis of the profile changes in fasting blood glucose, HbA1c, insulin, lipid panel and body composition, stratified by the Ala54Thr polymorphism. Our results show that the Thr54 allele carriers (those who were heterozygous or homozygous for the threonine-encoding allele had lower HDL cholesterol and higher triglyceride levels at baseline compared to the Ala54 homozygotes (those who were homozygous for the alanine-encoding allele. Both groups made clinically important improvements in lipid profiles and glycemic control as a response to the intervention. Whereas the Ala54 homozygotes decreased HDL cholesterol in the context of an overall total cholesterol decrease, Thr54 allele carriers increased HDL cholesterol as part of an overall total cholesterol decrease. We conclude that the Ala54Thr polymorphism of FABP2 modulates HDL cholesterol in Mexican-Americans with T2D and that Thr54 allele carriers may be responsive in interventions that include dietary changes.

A novel cell binding site in the coiled‐coil domain of laminin involved in capillary morphogenesis

DEFF Research Database (Denmark)

Sanz, Laura; García-Bermejo, Laura; Blanco, Francisco J

2003-01-01

Recently, we reported the isolation and characterization of an anti‐laminin antibody that modulates the extracellular matrix‐dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction....

Effect of anticonvulsant drugs on (35S)t-butylbicyclophosphorothionate binding in vitro and ex vivo

International Nuclear Information System (INIS)

Pitkaenen, A.; Riekkinen, P.J.; Saano, V.; Tuomisto, L.

1987-01-01

Using several concentrations of eight anticonvulsant drugs in clinical use (carbamazepine, clonazepam, phenytoin, phenobarbital, ethosuximide, primidone, sodium valproate, and D,L-γ-vinyl GABA), we studied their abilities in vitro to displace ( 35 S)t-butylbicyclophosphorothionate ( 35 S-TBPS) from its binding site in a homogenate of rat brain. Thereafter ethosuximide (150 mg/kg), phenobarbital (30 mg/kg), clonazepam (0.3 mg/kg), or phenytoin (100 mg/kg) was injected intraperitoneally into rats for 16-20 days; and the effect of drug administration on 35 S-TBPS binding was studied in the cortex and hippocampus ex vivo. Phenobarbital (100 μM, P 35 S-TBPS binding in vitro by 10-16%. After drug administration of phenobarbital (concentration in plasma 168 μM), the number of binding sites decreased and the binding affinity (p 35 S-TBPS binding in vitro at the concentration analogous to therapeutic plasma levels or ex vivo at the dose used. These results suggest that the use of phenobarbital may modulate the TBPS binding site, but the role of the present findings in the anticonvulsant action of phenobarbital needs to be further studied. (author)

Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

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Paulo M F Cunha

2010-07-01

Full Text Available Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2 or the zinc-finger transcription factor lame duck (lmd lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

Resveratrol modulates the inflammatory response via an estrogen receptor-signal integration network

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Nwachukwu, Jerome C; Srinivasan, Sathish; Bruno, Nelson E; Parent, Alexander A; Hughes, Travis S; Pollock, Julie A; Gjyshi, Olsi; Cavett, Valerie; Nowak, Jason; Garcia-Ordonez, Ruben D; Houtman, René; Griffin, Patrick R; Kojetin, Douglas J; Katzenellenbogen, John A; Conkright, Michael D; Nettles, Kendall W

2014-01-01

Resveratrol has beneficial effects on aging, inflammation and metabolism, which are thought to result from activation of the lysine deacetylase, sirtuin 1 (SIRT1), the cAMP pathway, or AMP-activated protein kinase. In this study, we report that resveratrol acts as a pathway-selective estrogen receptor-α (ERα) ligand to modulate the inflammatory response but not cell proliferation. A crystal structure of the ERα ligand-binding domain (LBD) as a complex with resveratrol revealed a unique perturbation of the coactivator-binding surface, consistent with an altered coregulator recruitment profile. Gene expression analyses revealed significant overlap of TNFα genes modulated by resveratrol and estradiol. Furthermore, the ability of resveratrol to suppress interleukin-6 transcription was shown to require ERα and several ERα coregulators, suggesting that ERα functions as a primary conduit for resveratrol activity. DOI: http://dx.doi.org/10.7554/eLife.02057.001 PMID:24771768

Dissecting stimulus-response binding effects: Grouping by color separately impacts integration and retrieval processes.

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Laub, Ruth; Frings, Christian; Moeller, Birte

2018-04-23

In selection tasks, target and distractor features can be encoded together with the response into the same short-lived memory trace, or event file (see Hommel, 2004), leading to bindings between stimulus and response features. The repetition of a stored target or distractor feature can lead to the retrieval of the entire episode, including the response-so-called "binding effects." Binding effects due to distractor repetition are stronger for grouped than for nongrouped target and distractor stimulus configurations. Modulation of either of two mechanisms that lead to the observed binding effects might be responsible here: Grouping may influence either stimulus-response integration or stimulus-response retrieval. In the present study we investigated the influences of grouping on both mechanisms independently. In two experiments, target and distractor letters were grouped (or nongrouped) via color (dis)similarity separately during integration and retrieval. Grouping by color similarity affected integration and retrieval mechanisms independently and in different ways. Color dissimilarity enhanced distractor-based retrieval, whereas color similarity enhanced distractor integration. We concluded that stimulus grouping is relevant for binding effects, but that the mechanisms that contribute to binding effects should be carefully separated.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

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Singh, Anirudh K; Woodiga, Shireen A; Grau, Margaret A; King, Samantha J

2017-03-01

Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis , like Streptococcus gordonii and Streptococcus sanguinis , binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. Copyright © 2017 American Society for Microbiology.

Tight binding simulation study on zigzag single-walled carbon nanotubes

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Sharma, Deepa; Jaggi, Neena; Gupta, Vishu

2018-01-01

Tight binding simulation studies using the density functional tight binding (DFTB) model have been performed on various zigzag single-walled carbon-nanotubes (SWCNTs) to investigate their electronic properties using DFTB module of the Material Studio Software version 7.0. Various combinations of different eigen-solvers and charge mixing schemes available in the DFTB Module have been tried to chalk out the electronic structure. The analytically deduced values of the bandgap of (9, 0) SWCNT were compared with the experimentally determined value reported in the literature. On comparison, it was found that the tight binding approximations tend to drastically underestimate the bandgap values. However, the combination of Anderson charge mixing method with standard eigensolver when implemented using the smart algorithm was found to produce fairly close results. These optimized model parameters were then used to determine the band structures of various zigzag SWCNTs. (9, 0) Single-walled Nanotube which is extensively being used for sensing NH3, CH4 and NO2 has been picked up as a reference material since its experimental bandgap value has been reported in the literature. It has been found to exhibit a finite energy bandgap in contrast to its expected metallic nature. The study is of utmost significance as it not only probes and validates the simulation route for predicting suitable properties of nanomaterials but also throws light on the comparative efficacy of the different approximation and rationalization quantum mechanical techniques used in simulation studies. Such simulation studies if used intelligently prove to be immensely useful to the material scientists as they not only save time and effort but also pave the way to new experiments by making valuable predictions.

Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

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Jason R Gerstner

2011-01-01

Full Text Available Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7 on sleep and long-term memory (LTM formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

DEFF Research Database (Denmark)

Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik

2012-01-01

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the a...... lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution....

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

Energy Technology Data Exchange (ETDEWEB)

Wada, Takeyoshi [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Asahi, Toru [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Sawamura, Naoya, E-mail: naoya.sawamura@gmail.com [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan)

2016-08-26

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

International Nuclear Information System (INIS)

Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

2016-01-01

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

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Daniel J White

Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

Small-molecule modulators of PXR and CAR

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Chai, Sergio C.; Cherian, Milu T.; Wang, Yue-Ming; Chen, Taosheng

2016-01-01

Two nuclear receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), participate in the xenobiotic detoxification system by regulating the expression of drug-metabolizing enzymes and transporters in order to degrade and excrete foreign chemicals or endogenous metabolites. This review aims to expand the perceived relevance of PXR and CAR beyond their established role as master xenosensors to disease-oriented areas, emphasizing their modulation by small molecules. Structural studies of these receptors have provided much-needed insight into the nature of their binding promiscuity and the important elements that lead to ligand binding. Reports of species- and isoform-selective activation highlight the need for further scrutiny when extrapolating from animal data to humans, as animal models are at the forefront of early drug discovery. PMID:26921498

Vitamin D binding protein: a multifunctional protein of clinical importance.

Science.gov (United States)

Speeckaert, Marijn M; Speeckaert, Reinhart; van Geel, Nanja; Delanghe, Joris R

2014-01-01

Since the discovery of group-specific component and its polymorphism by Hirschfeld in 1959, research has put spotlight on this multifunctional transport protein (vitamin D binding protein, DBP). Besides the transport of vitamin D metabolites, DBP is a plasma glycoprotein with many important functions, including sequestration of actin, modulation of immune and inflammatory responses, binding of fatty acids, and control of bone development. A considerable DBP polymorphism has been described with a specific allele distribution in different geographic area. Multiple studies have shed light on the interesting relationship between polymorphisms of the DBP gene and the susceptibility to diseases. In this review, we give an overview of the multifunctional character of DBP and describe the clinical importance of DBP and its polymorphisms. Finally, we discuss the possibilities to use DBP as a novel therapeutic agent.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

Science.gov (United States)

Barreales, Eva G; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier; Aparicio, Jesús F

2018-05-15

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR SARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding. IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is

Location-Unbound Color-Shape Binding Representations in Visual Working Memory.

Science.gov (United States)

Saiki, Jun

2016-02-01

The mechanism by which nonspatial features, such as color and shape, are bound in visual working memory, and the role of those features' location in their binding, remains unknown. In the current study, I modified a redundancy-gain paradigm to investigate these issues. A set of features was presented in a two-object memory display, followed by a single object probe. Participants judged whether the probe contained any features of the memory display, regardless of its location. Response time distributions revealed feature coactivation only when both features of a single object in the memory display appeared together in the probe, regardless of the response time benefit from the probe and memory objects sharing the same location. This finding suggests that a shared location is necessary in the formation of bound representations but unnecessary in their maintenance. Electroencephalography data showed that amplitude modulations reflecting location-unbound feature coactivation were different from those reflecting the location-sharing benefit, consistent with the behavioral finding that feature-location binding is unnecessary in the maintenance of color-shape binding. © The Author(s) 2015.

Functional connectivity supporting the selective maintenance of feature-location binding in visual working memory

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Sachiko eTakahama

2014-06-01

Full Text Available Information on an object’s features bound to its location is very important for maintaining object representations in visual working memory. Interactions with dynamic multi-dimensional objects in an external environment require complex cognitive control, including the selective maintenance of feature-location binding. Here, we used event-related functional magnetic resonance imaging to investigate brain activity and functional connectivity related to the maintenance of complex feature-location binding. Participants were required to detect task-relevant changes in feature-location binding between objects defined by color, orientation, and location. We compared a complex binding task requiring complex feature-location binding (color-orientation-location with a simple binding task in which simple feature-location binding, such as color-location, was task-relevant and the other feature was task-irrelevant. Univariate analyses showed that the dorsolateral prefrontal cortex (DLPFC, hippocampus, and frontoparietal network were activated during the maintenance of complex feature-location binding. Functional connectivity analyses indicated cooperation between the inferior precentral sulcus (infPreCS, DLPFC, and hippocampus during the maintenance of complex feature-location binding. In contrast, the connectivity for the spatial updating of simple feature-location binding determined by reanalyzing the data from Takahama et al. (2010 demonstrated that the superior parietal lobule (SPL cooperated with the DLPFC and hippocampus. These results suggest that the connectivity for complex feature-location binding does not simply reflect general memory load and that the DLPFC and hippocampus flexibly modulate the dorsal frontoparietal network, depending on the task requirements, with the infPreCS involved in the maintenance of complex feature-location binding and the SPL involved in the spatial updating of simple feature-location binding.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

DEFF Research Database (Denmark)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch

2015-01-01

) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced...... to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4...

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

Energy Technology Data Exchange (ETDEWEB)

Botcheva K.; McCorkle S. R.; McCombie W. R.; Dunn J. J.; Anderson C. W.

2011-12-15

We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

[3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.

Science.gov (United States)

Yagle, Monica A; Martin, Michael W; de Fiebre, Christopher M; de Fiebre, NancyEllen C; Drewe, John A; Dillon, Glenn H

2003-12-01

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.

Science.gov (United States)

Xia, Pengpeng; Wang, Yiting; Zhu, Congrui; Zou, Yajie; Yang, Ying; Liu, Wei; Hardwidge, Philip R; Zhu, Guoqiang

2016-02-09

F4(+) enterotoxigenic Escherichia coli (ETEC) strains cause diarrheal disease in neonatal and post-weaned piglets. Several different host receptors for F4 fimbriae have been described, with porcine aminopeptidase N (APN) reported most recently. The FaeG subunit is essential for the binding of the three F4 variants to host cells. Here we show in both yeast two-hybrid and pulldown assays that APN binds directly to FaeG, the major subunit of F4 fimbriae, from three serotypes of F4(+) ETEC. Modulating APN gene expression in IPEC-J2 cells affected ETEC adherence. Antibodies raised against APN or F4 fimbriae both reduced ETEC adherence. Thus, APN mediates the attachment of F4(+) E. coli to intestinal epithelial cells.

C2 Domains as Protein-Protein Interaction Modules in the Ciliary Transition Zone

Directory of Open Access Journals (Sweden)

Kim Remans

2014-07-01

Full Text Available RPGR-interacting protein 1 (RPGRIP1 is mutated in the eye disease Leber congenital amaurosis (LCA and its structural homolog, RPGRIP1-like (RPGRIP1L, is mutated in many different ciliopathies. Both are multidomain proteins that are predicted to interact with retinitis pigmentosa G-protein regulator (RPGR. RPGR is mutated in X-linked retinitis pigmentosa and is located in photoreceptors and primary cilia. We solved the crystal structure of the complex between the RPGR-interacting domain (RID of RPGRIP1 and RPGR and demonstrate that RPGRIP1L binds to RPGR similarly. RPGRIP1 binding to RPGR affects the interaction with PDEδ, the cargo shuttling factor for prenylated ciliary proteins. RPGRIP1-RID is a C2 domain with a canonical β sandwich structure that does not bind Ca2+ and/or phospholipids and thus constitutes a unique type of protein-protein interaction module. Judging from the large number of C2 domains in most of the ciliary transition zone proteins identified thus far, the structure presented here seems to constitute a cilia-specific module that is present in multiprotein transition zone complexes.

Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence

Directory of Open Access Journals (Sweden)

Gama Miguel

2008-10-01

Full Text Available Abstract Background Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD. The RGD sequence, present in several proteins from the extra-cellular matrix (ECM, is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an α-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. Results The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. Conclusion The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.

Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

International Nuclear Information System (INIS)

Poat, J.A.; Cripps, H.E.; Iversen, L.L.

1988-01-01

Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

Imidazoles and benzimidazoles as tubulin-modulators for anti-cancer therapy.

Science.gov (United States)

Torres, Fernando C; García-Rubiño, M Eugenia; Lozano-López, César; Kawano, Daniel F; Eifler-Lima, Vera L; von Poser, Gilsane L; Campos, Joaquín M

2015-01-01

Imidazoles and benzimidazoles are privileged heterocyclic bioactive compounds used with success in the clinical practice of innumerous diseases. Although there are many advancements in cancer therapy, microtubules remain as one of the few macromolecular targets validated for planning active anti-cancer compounds, and the design of drugs that modulate microtubule dynamics in unknown sites of tubulin is one of the goals of the medicinal chemistry. The discussion of the role of new and commercially available imidazole and benzimidazole derivatives as tubulin modulators is scattered throughout scientific literature, and indicates that these compounds have a tubulin modulation mechanism different from that of tubulin modulators clinically available, such as paclitaxel, docetaxel, vincristine and vinblastine. In fact, recent literature indicates that these derivatives inhibit microtubule formation binding to the colchicine site, present good pharmacokinetic properties and are capable of overcoming multidrug resistance in many cell lines. The understanding of the mechanisms involved in the imidazoles/benzimidazoles modulation of microtubule dynamics is very important to develop new strategies to overcome the resistance to anti-cancer drugs and to discover new biomarkers and targets for cancer chemotherapy.

Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin

DEFF Research Database (Denmark)

Enkavi, Giray; Mikkolainen, Heikki; Güngör, Burçin

2017-01-01

remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic......Niemann-Pick Protein C2 (npc2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc2-membrane interactions has......). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate (bmp) is specifically required...

A fractal analysis of protein to DNA binding kinetics using biosensors.

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Sadana, Ajit

2003-08-01

A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.

Specific binding of prostaglandin E2 to membrane preparations from human skin: receptor modulation by UVB-irradiation and chemical agents

International Nuclear Information System (INIS)

Lord, J.T.; Ziboh, V.A.

1979-01-01

Human skin membranes bind prostaglandin E2 (PGE2) with high affinity and specificity. This binding is inhibited by trypsin or heat treatment suggesting that PGE2 receptors have protein components. Exposure of the membranes to ultraviolet irradiation (UVB) resulted in the loss of the membrane binding capacity for PGE2. This UVB-inhibitory effect could be prevented by a known protein sulfhydryl-oxidizing agent and a known lipid anti-oxidant

Deciphering the BAR code of membrane modulators.

Science.gov (United States)

Salzer, Ulrich; Kostan, Julius; Djinović-Carugo, Kristina

2017-07-01

The BAR domain is the eponymous domain of the "BAR-domain protein superfamily", a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.

Different types of working memory binding in epilepsy patients with unilateral anterior temporal lobectomy.

Science.gov (United States)

van Geldorp, Bonnie; Bouman, Zita; Hendriks, Marc P H; Kessels, Roy P C

2014-03-01

The medial temporal lobe is an important structure for long-term memory formation, but its role in working memory is less clear. Recent studies have shown hippocampal involvement during working memory tasks requiring binding of information. It is yet unclear whether this is limited to tasks containing spatial features. The present study contrasted three binding conditions and one single-item condition in patients with unilateral anterior temporal lobectomy. A group of 43 patients with temporal lobectomy (23 left; 20 right) and 20 matched controls were examined with a working memory task assessing spatial relational binding (object-location), non-spatial relational binding (object-object), conjunctive binding (object-colour) and working memory for single items. We varied the delay period (3 or 6s), as there is evidence showing that delay length may modulate working memory performance. The results indicate that performance was worse for patients than for controls in both relational binding conditions, whereas patients were unimpaired in conjunctive binding. Single-item memory was found to be marginally impaired, due to a deficit on long-delay trials only. In conclusion, working memory binding deficits are found in patients with unilateral anterior temporal lobectomy. The role of the medial temporal lobe in working memory is not limited to tasks containing spatial features. Rather, it seems to be involved in relational binding, but not in conjunctive binding. The medial temporal lobe gets involved when working memory capacity does not suffice, for example when relations have to be maintained or when the delay period is long. Copyright © 2014 Elsevier Inc. All rights reserved.

Leptin Increases Striatal Dopamine D2 Receptor Binding in Leptin-Deficient Obese (ob/ob) Mice

Energy Technology Data Exchange (ETDEWEB)

Pfaffly, J.; Michaelides, M.; Wang, G-J.; Pessin, J.E.; Volkow, N.D.; Thanos, P.K.

2010-06-01

Peripheral and central leptin administration have been shown to mediate central dopamine (DA) signaling. Leptin-receptor deficient rodents show decreased DA D2 receptor (D2R) binding in striatum and unique DA profiles compared to controls. Leptin-deficient mice show increased DA activity in reward-related brain regions. The objective of this study was to examine whether basal D2R-binding differences contribute to the phenotypic behaviors of leptin-deficient ob/ob mice, and whether D2R binding is altered in response to peripheral leptin treatment in these mice. Leptin decreased body weight, food intake, and plasma insulin concentration in ob/ob mice but not in wild-type mice. Basal striatal D2R binding (measured with autoradiography [{sup 3}H] spiperone) did not differ between ob/ob and wild-type mice but the response to leptin did. In wild-type mice, leptin decreased striatal D2R binding, whereas, in ob/ob mice, leptin increased D2R binding. Our findings provide further evidence that leptin modulates D2R expression in striatum and that these effects are genotype/phenotype dependent.

Developments in South-East Asia

International Nuclear Information System (INIS)

Rahman, Reaz

1992-01-01

Interpretation of the Confidence-building and Confidence-building measures (CBMs) concerned with collective security, disarmament and arms control are treated for a specific case of Myanmar and Bangladesh. Political settings for confidence-building and constraints in Asia-Pacific region are defined including geopolitical imperatives, immediate strategic and internal imperatives as well as prospects for achievement of confidence-building. There is a growing agreement that CBMs should be modest, incremental and viewed as a process of cumulative accretion. On the global front, the continuing spread of unconventional weapons technologies, especially nuclear, chemical and biological, and advanced missile systems, remains a fundamental concern

Developments in South-East Asia

Energy Technology Data Exchange (ETDEWEB)

Rahman, Reaz [Ministry of Foreign Affairs, Dhaka (Bangladesh)

1993-12-31

Interpretation of the Confidence-building and Confidence-building measures (CBMs) concerned with collective security, disarmament and arms control are treated for a specific case of Myanmar and Bangladesh. Political settings for confidence-building and constraints in Asia-Pacific region are defined including geopolitical imperatives, immediate strategic and internal imperatives as well as prospects for achievement of confidence-building. There is a growing agreement that CBMs should be modest, incremental and viewed as a process of cumulative accretion. On the global front, the continuing spread of unconventional weapons technologies, especially nuclear, chemical and biological, and advanced missile systems, remains a fundamental concern

Applications of snake venom components to modulate integrin activities in cell-matrix interactions

Science.gov (United States)

Marcinkiewicz, Cezary

2013-01-01

Snake venom proteins are broadly investigated in the different areas of life science. Direct interaction of these compounds with cells may involve a variety of mechanisms that result in diverse cellular responses leading to the activation or blocking of physiological functions of the cell. In this review, the snake venom components interacting with integrins will be characterized in context of their effect on cellular response. Currently, two major families of snake venom proteins are considered as integrin-binding molecules. The most attention has been devoted to the disintegrin family, which binds certain types of integrins through specific motifs recognized as a tri-peptide structurally localized on an integrin-binding loop. Other snake venom integrin-binding proteins belong to the C-type lectin family. Snake venom molecules bind to the cellular integrins resulting in a modulation of cell signaling and in consequence, the regulation of cell proliferation, migration and apoptosis. Therefore, snake venom research on the integrin-binding molecules may have significance in biomedicine and basic cell biology. PMID:23811033

Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions

Energy Technology Data Exchange (ETDEWEB)

MacArthur, Stewart; Li, Xiao-Yong; Li, Jingyi; Brown, James B.; Chu, Hou Cheng; Zeng, Lucy; Grondona, Brandi P.; Hechmer, Aaron; Simirenko, Lisa; Keranen, Soile V.E.; Knowles, David W.; Stapleton, Mark; Bickel, Peter; Biggin, Mark D.; Eisen, Michael B.

2009-05-15

BACKGROUND: We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional. RESULTS: Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors. CONCLUSIONS: It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.

Development of allosteric modulators of GPCRs for treatment of CNS disorders.

Science.gov (United States)

Nickols, Hilary Highfield; Conn, P Jeffrey

2014-01-01

The discovery of allosteric modulators of G protein-coupled receptors (GPCRs) provides a promising new strategy with potential for developing novel treatments for a variety of central nervous system (CNS) disorders. Traditional drug discovery efforts targeting GPCRs have focused on developing ligands for orthosteric sites which bind endogenous ligands. Allosteric modulators target a site separate from the orthosteric site to modulate receptor function. These allosteric agents can either potentiate (positive allosteric modulator, PAM) or inhibit (negative allosteric modulator, NAM) the receptor response and often provide much greater subtype selectivity than orthosteric ligands for the same receptors. Experimental evidence has revealed more nuanced pharmacological modes of action of allosteric modulators, with some PAMs showing allosteric agonism in combination with positive allosteric modulation in response to endogenous ligand (ago-potentiators) as well as "bitopic" ligands that interact with both the allosteric and orthosteric sites. Drugs targeting the allosteric site allow for increased drug selectivity and potentially decreased adverse side effects. Promising evidence has demonstrated potential utility of a number of allosteric modulators of GPCRs in multiple CNS disorders, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as psychiatric or neurobehavioral diseases such as anxiety, schizophrenia, and addiction. © 2013.

Cucurbit[8]uril templated supramolecular ring structure formation and protein assembly modulation

NARCIS (Netherlands)

Ramaekers, M.; Wijnands, S.P.W.; van Dongen, J.L.J.; Brunsveld, L.; Dankers, P.Y.W.

2015-01-01

The interplay of Phe-Gly-Gly (FGG)-tagged proteins and bivalent FGG-tagged penta(ethylene glycol) as guest molecules with cucurbit[8]uril (Q8) hosts is studied to modulate the supramolecular assembly process. Ring structure formation of the bivalent guest molecule with Q8 leads to enhanced binding

Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

Science.gov (United States)

Chowdhury, Luvana; Croft, Celine J; Goel, Shikha; Zaman, Naina; Tai, Angela C-S; Walch, Erin M; Smith, Kelly; Page, Alexandra; Shea, Kevin M; Hall, C Dennis; Jishkariani, D; Pillai, Girinath G; Hall, Adam C

2016-06-01

GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 μM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the β3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Retinol-Binding Protein 4 and Insulin Resistance in Polycystic Ovary Syndrome

OpenAIRE

Hutchison, Samantha K.; Harrison, Cheryce; Stepto, Nigel; Meyer, Caroline; Teede, Helena J.

2008-01-01

OBJECTIVE?Polycystic ovary syndrome (PCOS) is an insulin-resistant state with insulin resistance being an established therapeutic target; however, measurement of insulin resistance remains challenging. We aimed to 1) determine serum retinol-binding protein 4 (RBP4) levels (purported to reflect insulin resistance) in women with PCOS and control subjects, 2) examine the relationship of RBP4 to conventional markers of insulin resistance, and 3) examine RBP4 changes with interventions modulating ...

Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details.

Science.gov (United States)

Rosenhouse-Dantsker, Avia

2018-01-01

In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84.

Science.gov (United States)

Mahmud, Zobaer Al; Jenkins, Laura; Ulven, Trond; Labéguère, Frédéric; Gosmini, Romain; De Vos, Steve; Hudson, Brian D; Tikhonova, Irina G; Milligan, Graeme

2017-12-20

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine 172 , located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [ 3 H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine 172 . These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.

A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

Science.gov (United States)

Henry, Kevin A.; Sulea, Traian; van Faassen, Henk; Hussack, Greg; Purisima, Enrico O.; MacKenzie, C. Roger; Arbabi-Ghahroudi, Mehdi

2016-01-01

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species. PMID:27631624

The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

Directory of Open Access Journals (Sweden)

Tochiki Keri K

2012-02-01

Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

2,3-DPG-Hb complex: a hypothesis for an asymmetric binding.

Science.gov (United States)

Pomponi, M; Bertonati, C; Fuglei, E; Wiig, O; Derocher, A E

2000-05-15

This study was undertaken to test the symmetry of 2,3-diphosphoglycerate (2,3-DPG) binding site in hemoglobin (Hb). From Arnone's study [A. Arnone, Nature (London) 237 (1972) 146] the 2,3-DPG binding site is located at the top of the cavity, that runs through the center of the deoxy-Hb molecule. However, it is possible that this symmetry reported by Arnone, for crystals of 2,3-DPG-Hb complex, might not be conserved in solution. In this paper, we report the 31P nuclear magnetic resonances of the 2,3-DPG interaction with Hb. The 2,3-DPG chemical shifts of the P2 and P3 resonance are both pH- and hemoglobin-dependent [protein from man, polar bear (Ursus maritimus), Arctic fox (Alopex lagopus) and bovine]. 2,3-DPG binds tightly to deoxyhemoglobin and weakly, nevertheless significantly, to oxyhemoglobin. In particular, our results suggest similar spatial position of the binding site of 2,3-DPG in both forms of Hb in solutions. However, the most unexpected result was the apparent loss of symmetry in the binding site, which might correlate with the ability of the hemoglobin to modulate its functional behavior. The different interactions of the phosphate groups indicate small differences in the quaternary structure of the different deoxy forms of hemoglobin. Given the above structural perturbation an asymmetric binding in the complex could justify, at least in part, different physiological properties of Hb. Regardless, functionally relevant effects of 2,3-DPG seem to be measured and best elucidated through solution studies.

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

Science.gov (United States)

Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

2003-01-01

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

An Experiment Illustrating the Change in Ligand p"K"[subscript a] upon Protein Binding

Science.gov (United States)

Chenprakhon, Pirom; Panijpan, Bhinyo; Chaiyen, Pimchai

2012-01-01

The modulation of ligand p"K"[subscript a] due to its surrounding environment is a crucial feature that controls many biological phenomena. For example, the shift in the p"K"[subscript a] of substrates or catalytic residues at enzyme active sites upon substrate binding often triggers and controls enzymatic reactions. In this work, we developed an…

Chemical shift assignments of the first and second RRMs of Nrd1, a fission yeast MAPK-target RNA binding protein.

Science.gov (United States)

Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

2017-10-01

Negative regulator differentiation 1 (Nrd1), a fission yeast RNA binding protein, modulates cytokinesis and sexual development and contributes to stress granule formation in response to environmental stresses. Nrd1 comprises four RRM domains and binds and stabilizes Cdc4 mRNA that encodes the myosin II light chain. Nrd1 binds the Cpc2 fission-yeast RACK1 homolog, and the interaction promotes Nrd1 localization to stress granules. Interestingly, Pmk1 mitogen-activated protein kinase phosphorylates Thr40 in the unstructured N-terminal region and Thr126 in the first RRM domain of Nrd1. Phosphorylation significantly reduces RNA-binding activity and likely modulates Nrd1 function. To reveal the relationship between the structure and function of Nrd1 and how phosphorylation affects structure, we used heteronuclear NMR techniques to investigate the three-dimensional structure of Nrd1. Here we report the 1 H, 13 C, and 15 N resonance assignments of RRM1-RRM2 (residues 108-284) comprising the first and second RRMs obtained using heteronuclear NMR techniques. Secondary structures derived from the chemical shifts are reported. These data should contribute to the understanding of the three-dimensional structure of the RRM1-RRM2 region of Nrd1 and the perturbation caused by phosphorylation.

New perspectives on mannan-binding lectin-mediated complement activation

DEFF Research Database (Denmark)

Degn, Søren Egedal; Thiel, Steffen; Jensenius, Jens Christian

2007-01-01

The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP......, allowing C3 activation in the absence of components otherwise believed critical. The classical bypass pathways are dependent on C1 and components of the AP. A recent study has shown the existence also of a lectin bypass pathway dependent on mannan-binding lectin (MBL) and AP components. The emerging...

An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation.

Science.gov (United States)

Sabbattini, Pierangela; Sjoberg, Marcela; Nikic, Svetlana; Frangini, Alberto; Holmqvist, Per-Henrik; Kunowska, Natalia; Carroll, Tom; Brookes, Emily; Arthur, Simon J; Pombo, Ana; Dillon, Niall

2014-03-01

Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

DEFF Research Database (Denmark)

1995-01-01

Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody

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Chiara Florio

2013-02-01

Full Text Available Palytoxin (PLTX is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA, a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 × 10−10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10−7 M and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10−9 M. Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10−5 M. However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

Autoradiographic evidence of sup 125 I-. beta. -endorphin binding sites in the articular cartilage of the rat

Energy Technology Data Exchange (ETDEWEB)

Castano, M.T.; Freire-Garabal, M.; Giraldez, M.; Nunez, M.J.; Belmonte, A.; Couceiro, J.; Jorge, J. (Univ. of Santiago (Spain))

1991-01-01

After {sup 125}I-{beta}-endorphin was intravenously injected to rats, an autoradiographic study of distal femur articular cartilage was performed. Results show a specific binding of {sup 125}I-{beta}-endorphin to chondrocytes, suggesting the possible existence of an opiate modulation of articular cartilage.

Dynamic in vivo binding of transcription factors to cis-regulatory modules of cer and gsc in the stepwise formation of the Spemann–Mangold organizer

Science.gov (United States)

Sudou, Norihiro; Yamamoto, Shinji; Ogino, Hajime; Taira, Masanori

2012-01-01

How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann–Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer. PMID:22492356

Dynamic in vivo binding of transcription factors to cis-regulatory modules of cer and gsc in the stepwise formation of the Spemann-Mangold organizer.

Science.gov (United States)

Sudou, Norihiro; Yamamoto, Shinji; Ogino, Hajime; Taira, Masanori

2012-05-01

How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann-Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.

Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

Science.gov (United States)

Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

2014-08-17

Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure. Copyright © 2014. Published by Elsevier Ireland Ltd.

The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

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Kratochwil Nicole A

2007-10-01

Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

Alignment and prediction of cis-regulatory modules based on a probabilistic model of evolution.

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Xin He

2009-03-01

Full Text Available Cross-species comparison has emerged as a powerful paradigm for predicting cis-regulatory modules (CRMs and understanding their evolution. The comparison requires reliable sequence alignment, which remains a challenging task for less conserved noncoding sequences. Furthermore, the existing models of DNA sequence evolution generally do not explicitly treat the special properties of CRM sequences. To address these limitations, we propose a model of CRM evolution that captures different modes of evolution of functional transcription factor binding sites (TFBSs and the background sequences. A particularly novel aspect of our work is a probabilistic model of gains and losses of TFBSs, a process being recognized as an important part of regulatory sequence evolution. We present a computational framework that uses this model to solve the problems of CRM alignment and prediction. Our alignment method is similar to existing methods of statistical alignment but uses the conserved binding sites to improve alignment. Our CRM prediction method deals with the inherent uncertainties of binding site annotations and sequence alignment in a probabilistic framework. In simulated as well as real data, we demonstrate that our program is able to improve both alignment and prediction of CRM sequences over several state-of-the-art methods. Finally, we used alignments produced by our program to study binding site conservation in genome-wide binding data of key transcription factors in the Drosophila blastoderm, with two intriguing results: (i the factor-bound sequences are under strong evolutionary constraints even if their neighboring genes are not expressed in the blastoderm and (ii binding sites in distal bound sequences (relative to transcription start sites tend to be more conserved than those in proximal regions. Our approach is implemented as software, EMMA (Evolutionary Model-based cis-regulatory Module Analysis, ready to be applied in a broad biological context.

Structure of a fibronectin type III-like module from Clostridium thermocellum

International Nuclear Information System (INIS)

Alahuhta, Markus; Xu, Qi; Brunecky, Roman; Adney, William S.; Ding, Shi-You; Himmel, Michael E.; Lunin, Vladimir V.

2010-01-01

The 1.6 Å resolution structure of a fibronectin type III-like module from Clostridium thermocellum with two molecules in the asymmetric unit is reported. The 1.6 Å resolution structure of a fibronectin type III-like module from Clostridium thermocellum with two molecules in the asymmetric unit is reported. The crystals used for data collection belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 35.43, b = 45.73, c = 107.72 Å, and the structure was refined to an R factor of 0.166. Structural comparisons found over 800 similar structures in the Protein Data Bank. The broad range of different proteins or protein domains with high structural similarity makes it especially demanding to classify these proteins. Previous studies of fibronectin type III-like modules have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules or as proteins that help large enzyme complexes remain soluble

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.

Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

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Shoni eBruell

2013-11-01

Full Text Available Relaxin family peptide (RXFP receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP based signalling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a

Cortisol decreases 2[125I] iodomelatonin binding sites in the duck thymus

International Nuclear Information System (INIS)

Poon, A.M.S.; Liu, Z.M.; Tang, F.; Pang, S.F.

1994-01-01

The immunosuppressive effect of chronic glucocorticoid treatment on 2[ 125 I] iodomelatonin binding in the duck thymus was studied. Two-week-old ducks were injected intraperitoneally with either 1 mg of cortisol per day (experimental group) or an equivalent volume of vehicle (control group) in the middle of the light period for seven days. 2[ 125 I] iodomelatonin binding assays were performed on thymic membranes. Cortisol injection reduced the body weight gain, size of the bursa of Fabricius and absolute weights of the primary lymphoid organs but had no effect on the spleen weights. The relative weights of the spleen were increased while those of the primary lymphoid organs were unchanged. The density of the thymus 2[ 125 I] iodomelatonin binding sites was decreased while the affinity was not affected. The modulation of the thymic 2[ 125 I] iodomelatonin binding sites by changes in the immune status of the duck suggests that these binding sites represent physiologically relevant melatonin receptors and that melatonin exerts its action on the lymphoid tissues directly. The authors findings support the hypothesis that the thymus is the target site for the immunomodulatory interactions between the pineal melatonin and the adrenal steroids. A possible inhibitory influence of adrenal steroids on the immuno-enhancing effect of melatonin is also suggested. 34 refs., 3 tabs

Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

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Down Thomas A

2010-09-01

Full Text Available Abstract Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS" but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq not to be biological transcription factor binding sites ("empirical TFBS". We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

Molecular pharmacology of cell receptors for cardiac glycosides, opiates, ACTH and ion channel modulators

Energy Technology Data Exchange (ETDEWEB)

Hnatowich, M R

1986-01-01

The influence of light and oxygen on molecular interactions between the artificial food dye, erythrosine (ERY), and (/sup 3/H)ouabain ((/sup 3/H)OUA) binding sites on (Na/sup +/ + K/sup +/)-ATPase in rat brain and guinea pig heart was investigated. Putative endogenous digitalis-like factors (DLF's) were studied in four in vitro assays for cardiac glycosides. (/sup 3/H)Etorphine binding was characterized in rat brain homogenates, depleted of opioids, from animals acutely and chronically treated with morphine and naloxone, and either unstressed or cold-restraint-stressed. Binding sites for the ion channel modulators (/sup 3/H)verapamil ((/sup 3/H)VER) and (/sup 3/H) phencyclidine ((/sup 3/H)PCP) were characterized in rat brain.

Allosteric Binding in the Serotonin Transporter - Pharmacology, Structure, Function and Potential Use as a Novel Drug Target

DEFF Research Database (Denmark)

Loland, Claus J.; Sanchez, Connie; Plenge, Per

2017-01-01

The serotonin transporter (SERT) is an important drug target and the majority of currently used antidepressants are potent inhibitors of SERT, binding primarily to the substrate binding site. However, even though the existence of an allosteric modulator site was realized more than 30 years ago......, the research into this mechanism is still in its early days. The current knowledge about the allosteric site with respect to pharmacology, structure and function, and pharmacological tool compounds, is reviewed and a perspective is given on its potential as a drug target....

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

DEFF Research Database (Denmark)

Joosten, I; Wauben, M H; Holewijn, M C

1994-01-01

New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC-peptide-TCR interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptides one...... characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides...

Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

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Marlet Martinez-Archundia

2012-01-01

Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.

Science.gov (United States)

Laverty, Duncan; Thomas, Philip; Field, Martin; Andersen, Ole J; Gold, Matthew G; Biggin, Philip C; Gielen, Marc; Smart, Trevor G

2017-11-01

γ-Aminobutyric acid receptors (GABA A Rs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABA A Rs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABA A R function requires high-resolution structures. Here we present the first atomic structures of a GABA A R chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABA A Rs in neurological disorders.

Binding of cetuximab to the EGFRvIII deletion mutant and its biological consequences in malignant glioma cells

International Nuclear Information System (INIS)

Jutten, Barry; Dubois, Ludwig; Li Younan; Aerts, Hugo; Wouters, Bradly G.; Lambin, Philippe; Theys, Jan; Lammering, Guido

2009-01-01

Background and purpose: Despite the clinical use of cetuximab, a chimeric antibody against EGFR, little is known regarding its interaction with EGFRvIII, a frequently expressed deletion mutant of EGFR. Therefore, we investigated the interaction and the functional consequences of cetuximab treatment on glioma cells stably expressing EGFRvIII. Materials and methods: The human glioma cell line U373 genetically modified to express EGFRvIII was used to measure the binding of cetuximab and its internalization using flow cytometry and confocal microscopy. Proliferation and cell survival were analyzed by cell growth and clonogenic survival assays. Results: Cetuximab is able to bind to EGFRvIII and causes an internalization of the receptor and decreases its expression levels. Furthermore, in contrast to EGF, cetuximab was able to activate EGFRvIII which was evidenced by multiple phosphorylation sites and its downstream signaling targets. Despite this activation, the growth rate and the radiosensitivity of the EGFRvIII-expressing glioma cells were not modulated. Conclusions: Cetuximab binds to EGFRvIII and leads to the initial activation, internalization and subsequent downregulation of EGFRvIII, but it does not seem to modulate the proliferation or radiosensitivity of EGFRvIII-expressing glioma cells. Thus, approaches to treat EGFRvIII-expressing glioma cells should be evaluated more carefully.

PReMod: a database of genome-wide mammalian cis-regulatory module predictions.

Science.gov (United States)

Ferretti, Vincent; Poitras, Christian; Bergeron, Dominique; Coulombe, Benoit; Robert, François; Blanchette, Mathieu

2007-01-01

We describe PReMod, a new database of genome-wide cis-regulatory module (CRM) predictions for both the human and the mouse genomes. The prediction algorithm, described previously in Blanchette et al. (2006) Genome Res., 16, 656-668, exploits the fact that many known CRMs are made of clusters of phylogenetically conserved and repeated transcription factors (TF) binding sites. Contrary to other existing databases, PReMod is not restricted to modules located proximal to genes, but in fact mostly contains distal predicted CRMs (pCRMs). Through its web interface, PReMod allows users to (i) identify pCRMs around a gene of interest; (ii) identify pCRMs that have binding sites for a given TF (or a set of TFs) or (iii) download the entire dataset for local analyses. Queries can also be refined by filtering for specific chromosomal regions, for specific regions relative to genes or for the presence of CpG islands. The output includes information about the binding sites predicted within the selected pCRMs, and a graphical display of their distribution within the pCRMs. It also provides a visual depiction of the chromosomal context of the selected pCRMs in terms of neighboring pCRMs and genes, all of which are linked to the UCSC Genome Browser and the NCBI. PReMod: http://genomequebec.mcgill.ca/PReMod.

Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Science.gov (United States)

Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity. PMID:23990797

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

Directory of Open Access Journals (Sweden)

Lifeng Liu

Full Text Available Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1, a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

Science.gov (United States)

Liu, Lifeng; Shang-Guan, Keke; Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease.

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Katrina Soderquest

2017-02-01

Full Text Available The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21 specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn's disease, ulcerative colitis (UC and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner.

KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1

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Borgel, Julie; Tyl, Marek; Schiller, Karin; Pusztai, Zsofia; Dooley, Christopher M.; Deng, Wen; Wooding, Carol; White, Richard J.; Warnecke, Tobias; Leonhardt, Heinrich; Busch-Nentwich, Elisabeth M.

2017-01-01

Abstract Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing. PMID:28180290

Theta Phase Synchronization Is the Glue that Binds Human Associative Memory.

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Clouter, Andrew; Shapiro, Kimron L; Hanslmayr, Simon

2017-10-23

Episodic memories are information-rich, often multisensory events that rely on binding different elements [1]. The elements that will constitute a memory episode are processed in specialized but distinct brain modules. The binding of these elements is most likely mediated by fast-acting long-term potentiation (LTP), which relies on the precise timing of neural activity [2]. Theta oscillations in the hippocampus orchestrate such timing as demonstrated by animal studies in vitro [3, 4] and in vivo [5, 6], suggesting a causal role of theta activity for the formation of complex memory episodes, but direct evidence from humans is missing. Here, we show that human episodic memory formation depends on phase synchrony between different sensory cortices at the theta frequency. By modulating the luminance of visual stimuli and the amplitude of auditory stimuli, we directly manipulated the degree of phase synchrony between visual and auditory cortices. Memory for sound-movie associations was significantly better when the stimuli were presented in phase compared to out of phase. This effect was specific to theta (4 Hz) and did not occur in slower (1.7 Hz) or faster (10.5 Hz) frequencies. These findings provide the first direct evidence that episodic memory formation in humans relies on a theta-specific synchronization mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Burn injury reveals altered phenotype in mannan-binding lectin-deficient mice

DEFF Research Database (Denmark)

Møller-Kristensen, Mette; Hamblin, Michael R; Thiel, Steffen

2007-01-01

Burn injury destroys skin, the second largest innate immune organ in the body, and triggers chaotic immune and inflammatory responses. The pattern recognition molecule, mannan-binding lectin (MBL), plays an important role in the first-line host defense against infectious agents. MBL initiates...... the lectin complement pathway and acts as an opsonin. Recent studies suggest that MBL also modulates inflammatory responses. We report that local responses after burn in MBL null mice differ from those found in wild-type (WT) mice in the following important biological markers: spontaneous eschar separation......, thinned epidermis and dermis, upregulation of soluble factors including cytokines, chemokines, cell adhesion molecules, a growth factor-binding protein, and matrix metalloproteinases. Mice lacking C1q, C4, or C3 did not show the lack of eschar separation seen in MBL null-burn phenotype. These findings...

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

Science.gov (United States)

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

The relationship between transcription initiation RNAs and CCCTC-binding factor (CTCF localization

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Taft Ryan J

2011-08-01

Full Text Available Abstract Background Transcription initiation RNAs (tiRNAs are nuclear localized 18 nucleotide RNAs derived from sequences immediately downstream of RNA polymerase II (RNAPII transcription start sites. Previous reports have shown that tiRNAs are intimately correlated with gene expression, RNA polymerase II binding and behaviors, and epigenetic marks associated with transcription initiation, but not elongation. Results In the present work, we show that tiRNAs are commonly found at genomic CCCTC-binding factor (CTCF binding sites in human and mouse, and that CTCF sites that colocalize with RNAPII are highly enriched for tiRNAs. To directly investigate the relationship between tiRNAs and CTCF we examined tiRNAs originating near the intronic CTCF binding site in the human tumor suppressor gene, p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A. Inhibition of CTCF-proximal tiRNAs resulted in increased CTCF localization and increased p21 expression, while overexpression of CTCF-proximal tiRNA mimics decreased CTCF localization and p21 expression. We also found that tiRNA-regulated CTCF binding influences the levels of trimethylated H3K27 at the alternate upstream p21 promoter, and affects the levels of alternate p21 (p21alt transcripts. Extending these studies to another randomly selected locus with conserved CTCF binding we found that depletion of tiRNA alters nucleosome density proximal to sites of tiRNA biogenesis. Conclusions Taken together, these data suggest that tiRNAs modulate local epigenetic structure, which in turn regulates CTCF localization.

Making Plans and Budgets Gender Responsive with CBMS: The ...

International Development Research Centre (IDRC) Digital Library (Canada)

Celia Reyes

Lack of disaggregated data on local socioeconomic conditions. • Insufficient knowledge on priority issues. / sectors. • Difficulty identifying individuals and households for targeted programs. • Very little knowledge of the socioeconomic and welfare status of the women and men in the localities. • Limited participation of other.

Selective androgen receptor modulators as function promoting therapies.

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Bhasin, Shalender; Jasuja, Ravi

2009-05-01

The past decade has witnessed an unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Although steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5alpha-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with androgen receptor (AR) contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand-binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein.

Science.gov (United States)

Sharadamma, N; Harshavardhana, Y; Singh, Pawan; Muniyappa, K

2010-06-01

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

Food deprivation modulates gamma-aminobutyric acid receptors and peripheral benzodiazepine binding sites in rats.

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Weizman, A; Bidder, M; Fares, F; Gavish, M

1990-12-03

The effect of 5 days of food deprivation followed by 5 days of refeeding on gamma-aminobutyric acid (GABA) receptors, central benzodiazepine receptors (CBR), and peripheral benzodiazepine binding sites (PBzS) was studied in female Sprague-Dawley rats. Starvation induced a decrease in the density of PBzS in peripheral organs: adrenal (35%; P less than 0.001), kidney (33%; P less than 0.01), and heart (34%; P less than 0.001). Restoration of [3H]PK 11195 binding to normal values was observed in all three organs after 5 days of refeeding. The density of PBzS in the ovary, pituitary, and hypothalamus was not affected by starvation. Food deprivation resulted in a 35% decrease in cerebellar GABA receptors (P less than 0.01), while CBR in the hypothalamus and cerebral cortex remained unaltered. The changes in PBzS observed in the heart and kidney may be related to the long-term metabolic stress associated with starvation and to the functional changes occurring in these organs. The down-regulation of the adrenal PBzS is attributable to the suppressive effect of hypercortisolemia on pituitary ACTH release. The reduction in cerebellar GABA receptors may be an adaptive response to food deprivation stress and may be relevant to the proaggressive effect of hunger.

SH2 domains: modulators of nonreceptor tyrosine kinase activity

OpenAIRE

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-01-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed ...

A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

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Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

2015-05-01

Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

Interplay of agency and ownership: the intentional binding and rubber hand illusion paradigm combined.

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Niclas Braun

Full Text Available The sense of agency (SoA refers to the phenomenal experience of initiating and controlling an action, whereas the sense of ownership (SoO describes the feeling of myness an agent experiences towards his or her own body parts. SoA has been investigated with intentional binding paradigms, and the sense of ownership (SoO with the rubber-hand illusion (RHI. We investigated the relationship between SoA and SoO by incorporating intentional binding into the RHI. Explicit and implicit measures of agency (SoA-questionnaire, intentional binding and ownership (SoO-questionnaire, proprioceptive drift were used. Artificial hand position (congruent/incongruent and mode of agent (self-agent/other-agent were systematically varied. Reported SoO varied mainly with position (higher in congruent conditions, but also with agent (higher in self-agent conditions. Reported SoA was modulated by agent (higher in self-agent conditions, and moderately by position (higher in congruent conditions. Implicit and explicit agency measures were not significantly correlated. Finally, intentional binding tended to be stronger in self-generated than observed voluntary actions. Results provide further evidence for a partial double dissociation between SoA and SoO, empirically distinct agency levels, and moderate intentional binding differences between self-generated and observed voluntary actions.

Interplay of agency and ownership: the intentional binding and rubber hand illusion paradigm combined.

Science.gov (United States)

Braun, Niclas; Thorne, Jeremy D; Hildebrandt, Helmut; Debener, Stefan

2014-01-01

The sense of agency (SoA) refers to the phenomenal experience of initiating and controlling an action, whereas the sense of ownership (SoO) describes the feeling of myness an agent experiences towards his or her own body parts. SoA has been investigated with intentional binding paradigms, and the sense of ownership (SoO) with the rubber-hand illusion (RHI). We investigated the relationship between SoA and SoO by incorporating intentional binding into the RHI. Explicit and implicit measures of agency (SoA-questionnaire, intentional binding) and ownership (SoO-questionnaire, proprioceptive drift) were used. Artificial hand position (congruent/incongruent) and mode of agent (self-agent/other-agent) were systematically varied. Reported SoO varied mainly with position (higher in congruent conditions), but also with agent (higher in self-agent conditions). Reported SoA was modulated by agent (higher in self-agent conditions), and moderately by position (higher in congruent conditions). Implicit and explicit agency measures were not significantly correlated. Finally, intentional binding tended to be stronger in self-generated than observed voluntary actions. Results provide further evidence for a partial double dissociation between SoA and SoO, empirically distinct agency levels, and moderate intentional binding differences between self-generated and observed voluntary actions.

Modulation of β-adrenergic receptors in the pituitary gland following adrenalectomy in rats

International Nuclear Information System (INIS)

Souza, E.B. de

1987-01-01

The effects of adrenalectomy on β-adrenergic receptors in the rat pituitary were examined using quantitative in vitro autoradiography with 125 I-iodocyanopindolol( 125 ICYP). 125 ICYP binding in the anterior, intermediate and posterior lobes of the pituitary gland was significantly increased in chronically adrenalectomized rats. The increase in 125 ICYP binding sites in the rat pituitary following adrenalectomy was not reversed by glucocorticoid replacement with dexamethasone. These data indicate that catecholamines of adrenomedullary origin are capable of modulating β-adrenergic receptors in the pituitary gland and suggest that peripheral epinephrine may be important in regulating pituitary hormone secretion. (author)

Resolving the fast kinetics of cooperative binding: Ca2+ buffering by calretinin.

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Guido C Faas

2007-11-01

Full Text Available Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity or increased (positive cooperativity. Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2

The ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering

Science.gov (United States)

Çamdere, Gamze; Guacci, Vincent; Stricklin, Jeremiah; Koshland, Douglas

2015-01-01

Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin’s DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition. DOI: http://dx.doi.org/10.7554/eLife.11315.001 PMID:26583750

Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

Science.gov (United States)

Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2014-03-18

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

DEFF Research Database (Denmark)

Helledie, T; Antonius, M; Sorensen, R V

2000-01-01

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty...

Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

Science.gov (United States)

Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

2016-03-14

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

Science.gov (United States)

Bethuyne, Jonas; De Gieter, Steven; Zwaenepoel, Olivier; Garcia-Pino, Abel; Durinck, Kaat; Verhelle, Adriaan; Hassanzadeh-Ghassabeh, Gholamreza; Speleman, Frank; Loris, Remy; Gettemans, Jan

2014-01-01

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations. PMID:25324313

Curcumin modulation of high fat diet-induced atherosclerosis and steatohepatosis in LDL receptor deficient mice

Science.gov (United States)

Consuming curcumin may benefit health by modulating lipid metabolism and suppressing atherogenesis. Fatty acid binding proteins (FABP-4/aP2) and CD36 expression are key factors in lipid accumulation in macrophages and foam cell formation in atherogenesis. Our earlier observations suggest that curcum...