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Sample records for calcium binding proteins

  1. Fractal aspects of calcium binding protein structures

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    Isvoran, Adriana [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania)], E-mail: aisvoran@cbg.uvt.ro; Pitulice, Laura [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania); Craescu, Constantin T. [INSERM U759/Institute Curie-Recherche, Centre Universitaire Paris-Sud, Batiment 112, 91405 Orsay (France); Chiriac, Adrian [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania)

    2008-03-15

    The structures of EF-hand calcium binding proteins may be classified into two distinct groups: extended and compact structures. In this paper we studied 20 different structures of calcium binding proteins using the fractal analysis. Nine structures show extended shapes, one is semi-compact and the other 10 have compact shapes. Our study reveals different fractal characteristics for protein backbones belonging to different structural classes and these observations may be correlated to the physicochemical forces governing the protein folding.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  3. Calcium-binding proteins from human platelets

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

    1983-06-01

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

  4. Apolipoprotein B is a calcium binding protein

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

    1986-05-29

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

  5. Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation.

    Science.gov (United States)

    Zhao, Y; Pokutta, S; Maurer, P; Lindt, M; Franklin, R M; Kappes, B

    1994-03-29

    Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.

  6. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

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    Craescu Constantin T

    2011-05-01

    Full Text Available Abstract Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.

  7. Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

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    Rosana L. Pagano

    2002-01-01

    Full Text Available Background: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice.

  8. Distribution of calcium-binding proteins in the chick visual system

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    C.P. Pfeiffer

    1997-11-01

    Full Text Available The calcium-binding proteins calbindin (CB, calretinin (CR, and parvalbumin (PV have been extensively studied over the last decade since they appear to be important as buffers of intracellular calcium. In the present study we investigated the distribution of these proteins in the chick visual system by means of conventional immunocytochemistry. The results indicated that CB, CR, and PV are widely distributed in retinorecipient areas of the chick brain. In some regions, all three calcium-binding proteins were present at different intensities and often in different neurons such as in the dorsolateral thalamic complex. In other areas, such as the nucleus geniculatus lateralis ventralis, only CB and CR were detected, whereas PV was absent. These results show that these three calcium-binding proteins are differentially distributed in the visual system of the chick, with varying degrees of co-localization

  9. The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

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    Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2014-10-13

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Cloning, purification, crystallization and preliminary crystallographic study of calcium-binding protein 5 from Entamoeba histolytica.

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    Kumar, Sanjeev; Zaidi, Rana; Gourinath, Samudrala

    2012-12-01

    Entamoeba histolytica is the causative agent of human amoebiasis. Phagocytosis is the major route of food intake by this parasite and is responsible for its virulence. Calcium and calcium-binding proteins play major roles in its phagocytosis. Calcium-binding protein 5 from E. histolytica (EhCaBP5) is a cytoplasmic protein; its expression is very sensitive to serum starvation and it seems to be involved in binding to myosin I. In this study, EhCaBP5 was cloned, expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. The purified protein crystallized in space group C222 and the crystals diffracted to 2 Å resolution. The Matthews coefficient indicated the presence of one molecule in the asymmetric unit, with a VM of 2.35 Å3 Da(-1) and a solvent content of 47.7%.

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

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

    2017-06-01

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

  12. Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

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    Xixi Cai

    2016-12-01

    Full Text Available Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives.

  13. Divergent action of calcium channel blockers on ATP-binding cassette protein expression.

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    Hasegawa, Kazuhiro; Wakino, Shu; Kanda, Takeshi; Yoshioka, Kyoko; Tatematsu, Satoru; Homma, Koichiro; Takamatsu, Ichiro; Sugano, Naoki; Hayashi, Koichi

    2005-12-01

    Calcium channel blockers (CCBs) are widely used in clinical practice, and have been reported to be effective in preventing the progression of atherosclerosis. We examined whether various types of calcium channel blockers affected the expression of ATP binding cassette transporter A1 (ABCA1), a factor contributing to anti-atherogenesis. Undifferentiated monocytic cell line, THP-1 cells were maintained in RPMI 1640 medium and treated with different kinds of calcium channel blockers. Among the calcium channel blockers tested, aranidipine and efonidipine increased ABCA1 protein expression without an increase in ABCA1 mRNA expression, whereas other calcium channel blockers (eg, nifedipine, amlodipine, and nicardipine) or T-type calcium channel blockers (eg, mibefradil and nickel chloride) failed to upregulate ABCA1 expression. H89, a protein kinase A inhibitor inhibited the aranidipine-induced ABCA1 protein expression, whereas genistein (a tyrosine kinase inhibitor), or AG490 (a JAK-2 inhibitor) had no effects. Neither of these inhibitors suppressed the efonidipine-induced ABCA1 protein expression. Intracellular cAMP levels were elevated only by aranidipine, but not by efonidipine. In conclusion, aranidipine and efonidipine have the ability to induce ABCA1 protein by distinct mechanisms; protein kinase A is involved in the aranidipine-induced ABCA1 upregulation. This non-class effect of calcium channel blockers may potentially offer beneficial action in the treatment of hypertensive subjects with atherosclerosis.

  14. Portraying the Effect of Calcium-Binding Proteins on Cytosolic Calcium Concentration Distribution Fractionally in Nerve Cells.

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    Jha, Brajesh Kumar; Joshi, Hardik; Dave, Devanshi D

    2016-11-23

    Nerve cells like neurons and astrocytes in central nervous system (CNS) take part in the signaling process which means the transformation of the information from one cell to another via signals. The signaling process is affected by various external parameters like buffers calcium-binding proteins, voltage-gated calcium channel. In the present paper, the role of buffers in the cytoplasmic calcium concentration distribution is shown. The elicitation in calcium concentration is due to the presence of lower amount calcium-binding proteins which can be shown graphically. The mathematical model is designed by keeping in mind the physiological condition taking place in CNS of mammalian brain. The thing to be noted here is that the more elicitation in the calcium concentration distribution results in the cell death which finally give neurodegenerative disease to the mammalian brain. The present paper gives a glimpse of Parkinson's diseases in particular. Computational results are performed in Wolfram Mathematica 9.0 and simulated on core(TM) i5-3210M CPU @ 2.50 GHz processing speed and 4 GB memory. It is found that the different types of buffer like ethylene glycol-bis([Formula: see text]-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and calmodulin have noteworthy effect at different fractions of time.

  15. Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design.

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    Slade, Daniel J; Fang, Pengfei; Dreyton, Christina J; Zhang, Ying; Fuhrmann, Jakob; Rempel, Don; Bax, Benjamin D; Coonrod, Scott A; Lewis, Huw D; Guo, Min; Gross, Michael L; Thompson, Paul R

    2015-04-17

    Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.

  16. Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton.

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    Webb, P D; Mahadevan, L C

    1987-12-18

    Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.

  17. Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen.

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    Bonura, A; Gulino, L; Trapani, A; Di Felice, G; Tinghino, R; Amoroso, S; Geraci, D; Valenta, R; Westritschnig, K; Scala, E; Mari, A; Colombo, P

    2008-05-01

    The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

  18. Identification of calcium-binding proteins associated with the human sperm plasma membrane

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    Westbrook Anne

    2010-01-01

    Full Text Available Abstract Background The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane. Methods Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface. Results Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1, HSPA5 (Bip, HYOU1 (ORP150, serum amyloid P-component (SAP and protein kinase C substrate 80K-H (80K-H were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm. Conclusion The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.

  19. APF/CBP, the small, amphipathic, anionic protein(s) in bile and gallstones, consists of lipid-binding and calcium-binding forms

    NARCIS (Netherlands)

    Lafont, H.; Domingo, N.; Groen, A.; Kaler, E. W.; Lee, S. P.; Koehler, R.; Ostrow, J. D.; Veis, A.

    1997-01-01

    Two very similar small anionic, amphipathic proteins, a phospholipid-binding apoprotein (anionic polypeptide fraction [APF]) and a calcium-binding polypeptide (CBP), are found abundantly in bile and all types of gallstones. The often disparate properties among various preparations of APF/CBP could

  20. Structural characterization of amorphous calcium carbonate-binding protein: an insight into the mechanism of amorphous calcium carbonate formation.

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    Su, Jingtan; Liang, Xiao; Zhou, Qiang; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2013-07-15

    ACC (amorphous calcium carbonate) plays an important role in biomineralization process for its function as a precursor for calcium carbonate biominerals. However, it is unclear how biomacromolecules regulate the formation of ACC precursor in vivo. In the present study, we used biochemical experiments coupled with bioinformatics approaches to explore the mechanisms of ACC formation controlled by ACCBP (ACC-binding protein). Size-exclusion chromatography, chemical cross-linking experiments and negative staining electron microscopy reveal that ACCBP is a decamer composed of two adjacent pentamers. Sequence analyses and fluorescence quenching results indicate that ACCBP contains two Ca²⁺-binding sites. The results of in vitro crystallization experiments suggest that one Ca²⁺-binding site is critical for ACC formation and the other site affects the ACC induction efficiency. Homology modelling demonstrates that the Ca²⁺-binding sites of pentameric ACCBP are arranged in a 5-fold symmetry, which is the structural basis for ACC formation. To the best of our knowledge, this is the first report on the structural basis for protein-induced ACC formation and it will significantly improve our understanding of the amorphous precursor pathway.

  1. The calcium binding protein ALG-2 binds and stabilizes Scotin, a p53-inducible gene product localized at the endoplasmic reticulum membrane

    DEFF Research Database (Denmark)

    Draeby, Ingrid; Woods, Yvonne L; la Cour, Jonas Marstrand

    2007-01-01

    ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG...

  2. Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

    DEFF Research Database (Denmark)

    Castillo, M B; Berchtold, M W; Rülicke, T

    1997-01-01

    To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

  3. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties

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    Xixi Cai

    2017-03-01

    Full Text Available Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

  4. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

    Science.gov (United States)

    Cai, Xixi; Yang, Qian; Lin, Jiaping; Fu, Nanyan; Wang, Shaoyun

    2017-03-29

    Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

  5. Calcium-binding proteins in the laterodorsal thalamic nucleus during development of the guinea pig.

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    Zakowski, Witold; Bogus-Nowakowska, Krystyna; Wasilewska, Barbara; Hermanowicz, Beata; Robak, Anna

    2014-11-01

    The laterodorsal thalamic nucleus (LD) is often treated as a part of the anterior thalamic nuclei (ATN) because of its location and similar connectivity. Our previous studies have shown that distribution of three calcium-binding proteins, i.e. calbindin D28k (CB), calretinin (CR) and parvalbumin (PV), changes within the ATN during development of the guinea pig. The aim of this study is to examine the immunoreactivity pattern of these proteins in the LD in the guinea pig ontogeny. Brains from animals ranging from 40th embryonic day to 80th postnatal day were used in the study. Two methods were applied: a single-labelling immunoenzymatic method and double-labelling immunofluorescence. No changes of the distribution pattern of the substances were observed throughout the examined developmental stages. CB and CR were the most abundantly expressed proteins in perikarya of the LD. Numerous CB- and CR-immunoreactive cell bodies were found throughout the whole extent of the nucleus. In most of these cell bodies both proteins colocalized vastly. The highest immunoreactivity of the perikarya containing CB and CR was observed in the mediodorsal part of the LD and in its rostral portion. In regard to PV, single cell bodies were observed mostly in the dorsal part of the nucleus. PV did not colocalize with the other proteins. In summary, all the studied calcium-binding proteins were already present in the LD at prenatal developmental stages and the pattern of distribution remained virtually constant until adulthood. Thus, the LD differs considerably from the ATN in an aspect of neurochemical cell differentiation. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.

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    Duplat, D; Puisségur, M; Bédouet, L; Rousseau, M; Boulzaguet, H; Milet, C; Sellos, D; Van Wormhoudt, A; Lopez, E

    2006-05-01

    Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95-99% calcium carbonate and 1-5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.

  7. Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus.

    Science.gov (United States)

    Leyrat, Cedric; Renner, Max; Harlos, Karl; Huiskonen, Juha T; Grimes, Jonathan M

    2014-01-07

    The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca²⁺ binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca²⁺ binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. 1H, 13C and 15N NMR assignments of a calcium-binding protein from Entamoeba histolytica.

    Science.gov (United States)

    Verma, Deepshikha; Bhattacharya, Alok; Chary, Kandala V R

    2016-04-01

    We report almost complete sequence specific (1)H, (13)C and (15)N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization.

  9. Distribution and Morphology of Calcium-Binding Proteins Immunoreactive Neurons following Chronic Tungsten Multielectrode Implants.

    Directory of Open Access Journals (Sweden)

    Marco Aurelio M Freire

    Full Text Available The development of therapeutic approaches to improve the life quality of people suffering from different types of body paralysis is a current major medical challenge. Brain-machine interface (BMI can potentially help reestablishing lost sensory and motor functions, allowing patients to use their own brain activity to restore sensorimotor control of paralyzed body parts. Chronic implants of multielectrodes, employed to record neural activity directly from the brain parenchyma, constitute the fundamental component of a BMI. However, before this technique may be effectively available to human clinical trials, it is essential to characterize its long-term impact on the nervous tissue in animal models. In the present study we evaluated how chronic implanted tungsten microelectrode arrays impact the distribution and morphology of interneurons reactive to calcium-binding proteins calbindin (CB, calretinin (CR and parvalbumin (PV across the rat's motor cortex. Our results revealed that chronic microelectrode arrays were well tolerated by the nervous tissue, with recordings remaining viable for up to 6 months after implantation. Furthermore, neither the morphology nor the distribution of inhibitory neurons were broadly impacted. Moreover, restricted microglial activation was observed on the implanted sites. On the whole, our results confirm and expand the notion that tungsten multielectrodes can be deemed as a feasible candidate to future human BMI studies.

  10. Auditory hindbrain atrophy and anomalous calcium binding protein expression after neonatal exposure to monosodium glutamate.

    Science.gov (United States)

    Foran, Lindsey; Blackburn, Kaitlyn; Kulesza, Randy J

    2017-03-06

    Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and is stored and released by both neurons and astrocytes. Despite the important role of glutamate as a neurotransmitter, elevated extracellular glutamate can result in excitotoxicity and apoptosis. Monosodium glutamate (MSG) is a naturally occurring sodium salt of glutamic acid that is used as a flavor enhancer in many processed foods. Previous studies have shown that MSG administration during the early postnatal period results in neurodegenerative changes in several forebrain regions, characterized by neuronal loss and neuroendocrine abnormalities. Systemic delivery of MSG during the neonatal period and induction of glutamate neurotoxicity in the cochlea have both been shown to result in fewer neurons in the spiral ganglion. We hypothesized that an MSG-induced loss of neurons in the spiral ganglion would have a significant impact on the number of neurons in the cochlear nuclei and superior olivary complex (SOC). Indeed, we found that exposure to MSG from postnatal days 4 through 10 resulted in significantly fewer neurons in the cochlear nuclei and SOC and significant dysmorphology in surviving neurons. Moreover, we found that neonatal MSG exposure resulted in a significant decrease in the expression of both calretinin and calbindin. These results suggest that neonatal exposure to MSG interferes with early development of the auditory brainstem and impacts expression of calcium binding proteins, both of which may lead to diminished auditory function. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Scaling properties of the radius of gyration and surface area for EF-hand calcium binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Pitulice, L. [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania); Isvoran, A. [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania)], E-mail: aisvoran@cbg.uvt.ro; Craescu, C.T. [INSERM U759/Institute Curie-Recherche, Centre Universitaire Paris-Sud, Batiment 112, 91405 Orsay (France); Chiriac, A. [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania)

    2009-04-30

    In this paper, we analyze the scaling properties of both the radius of gyration and the surface area for EF-hand calcium binding proteins. These properties are different for two conformational subfamilies: proteins with extended and compact structures, respectively. The radius of gyration is a measure of the shape of protein, whereas its surface fractal dimension is a measure of its interatomic packing. Different scaling properties for the radius of gyration underline that these two subfamilies present different shapes whilst different scaling properties for the surface area reveal different strengths of their intermolecular forces. All these data suggest different mechanisms responsible for the global folding of proteins belonging to these two subfamilies.

  12. Chitinase III in pomegranate seeds (Punica granatum Linn.): a high-capacity calcium-binding protein in amyloplasts.

    Science.gov (United States)

    Yang, Haixia; Zhang, Tuo; Masuda, Taro; Lv, Chenyan; Sun, Lei; Qu, Guiqin; Zhao, Guanghua

    2011-12-01

    Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  13. The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

    Science.gov (United States)

    Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

    2017-10-13

    Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that MAPK1-interacting and spindle-stabilizing (MISS)-like (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of secreted alkaline phosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Expression of the calcium-binding proteins MRP8 and MRP14 in monocytes is regulated by a calcium-induced suppressor mechanism.

    OpenAIRE

    ROTH, J.; Goebeler, M; Wrocklage, V; van den Bos, C; Sorg, C.

    1994-01-01

    MRP8 and MRP14 are two calcium-binding proteins of the S-100 family the expression of which is restricted to distinct stages of monocytic differentiation. Heteromeric MRP8/MRP14 complexes have been shown to represent their biologically active forms. However, it is not as yet clear whether biochemical modification of complexes, or regulation on the transcriptional level, are responsible for the control of MRP8/MRP14 expression. Employing Western-blot analysis and metabolic labelling we have de...

  15. The calcium-binding protein of Entamoeba histolytica as a fusion partner for expression of peptides in Escherichia coli.

    Science.gov (United States)

    Reddi, Honey; Bhattacharya, Alok; Kumar, Vijay

    2002-12-01

    We describe the construction of an Escherichia coli expression vector, CBP that allows the C-terminal fusion of heterologous proteins to the calcium-binding protein (CaBP) of the parasitic protozoan Entamoeba histolytica. The intrinsic nature of this protein to remain soluble on heat treatment has been exploited in its use as a novel fusion partner. The presence of a histidine tag and an enterokinase recognition site, aid in the affinity purification and proteolytic cleavage of the fusion protein. The efficacy of the vector was tested using the preS1 region of the envelope protein of the hepatitis B virus. The CaBP-preS1 fusion protein partitioned in the soluble fraction on heat treatment and this facilitated its rapid purification.

  16. Curcumin specifically binds to the human calcium-calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies.

    Science.gov (United States)

    Hoda, Nasimul; Naz, Huma; Jameel, Ehtesham; Shandilya, Ashutosh; Dey, Sharmistha; Hassan, Md Imtaiyaz; Ahmad, Faizan; Jayaram, B

    2016-01-01

    Calcium-calmodulin-dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus, it is involved in varieties of cellular functions such as cell signaling and neuronal survival. On the other hand, curcumin, a naturally occurring yellow bioactive component of turmeric possesses wide spectrum of biological actions, and it is widely used to treat atherosclerosis, diabetes, cancer, and inflammation. It also acts as an antioxidant. Here, we studied the interaction of curcumin with human CAMK4 at pH 7.4 using molecular docking, molecular dynamics (MD) simulations, fluorescence binding, and surface plasmon resonance (SPR) methods. We performed MD simulations for both neutral and anionic forms of CAMK4-curcumin complexes for a reasonably long time (150 ns) to see the overall stability of the protein-ligand complex. Molecular docking studies revealed that the curcumin binds in the large hydrophobic cavity of kinase domain of CAMK4 through several hydrophobic and hydrogen-bonded interactions. Additionally, MD simulations studies contributed in understanding the stability of protein-ligand complex system in aqueous solution and conformational changes in the CAMK4 upon binding of curcumin. A significant increase in the fluorescence intensity at 495 nm was observed (λexc = 425 nm), suggesting a strong interaction of curcumin to the CAMK4. A high binding affinity (KD = 3.7 × 10(-8) ± .03 M) of curcumin for the CAMK4 was measured by SPR further indicating curcumin as a potential ligand for the CAMK4. This study will provide insights into designing a new inspired curcumin derivatives as therapeutic agents against many life-threatening diseases.

  17. Short-term exposure to L-type calcium channel blocker, verapamil, alters the expression pattern of calcium-binding proteins in the brain of goldfish, Carassius auratus.

    Science.gov (United States)

    Palande, Nikhil V; Bhoyar, Rahul C; Biswas, Saikat P; Jadhao, Arun G

    2015-01-01

    The influx of calcium ions (Ca(2+)) is responsible for various physiological events including neurotransmitter release and synaptic modulation. The L-type voltage dependent calcium channels (L-type VDCCs) transport Ca(2+) across the membrane. Calcium-binding proteins (CaBPs) bind free cytosolic Ca(2+) and prevent excitotoxicity caused by sudden increase in cytoplasmic Ca(2+). The present study was aimed to understand the regulation of expression of neuronal CaBPs, namely, calretinin (CR) and parvalbumin (PV) following blockade of L-type VDCCs in the CNS of Carassius auratus. Verapamil (VRP), a potent L-type VDCC blocker, selectively blocks Ca(2+) entry at the plasma membrane level. VRP present in the aquatic environment at a very low residual concentration has shown ecotoxicological effects on aquatic animals. Following acute exposure for 96h, median lethal concentration (LC50) for VRP was found to be 1.22mg/L for goldfish. At various doses of VRP, the behavioral alterations were observed in the form of respiratory difficulty and loss of body balance confirming the cardiovascular toxicity caused by VRP at higher doses. In addition to affecting the cardiovascular system, VRP also showed effects on the nervous system in the form of altered expression of PV. When compared with controls, the pattern of CR expression did not show any variations, while PV expression showed significant alterations in few neuronal populations such as the pretectal nucleus, inferior lobes, and the rostral corpus cerebellum. Our result suggests possible regulatory effect of calcium channel blockers on the expression of PV. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Relating form and function of EF-hand calcium binding proteins.

    Science.gov (United States)

    Chazin, Walter J

    2011-03-15

    The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in

  19. Abnormal neuronal expression of the calcium-binding proteins, parvalbumin and calbindin D-28k, in aged dogs.

    Science.gov (United States)

    Sisó, S; Tort, S; Aparici, C; Pérez, L; Vidal, E; Pumarola, M

    2003-01-01

    Disturbances of the gamma-aminobutyric acid (GABA) neurotransmitter system have been implicated in chronic degenerative neurological disease. Cognitive dysfunction and neuron loss are features in older dogs. GABAergic neurons also show immunoreactivity for specific calcium-binding proteins. Immunohistochemistry was used to study the neuronal expression of calbindin D-28k and parvalbumin in different areas of the brain in 13 dogs, aged between 2 and 13.5 years. Calbindin expression was found only in the cerebellum. There were significant differences in the quantity and distribution of neurons expressing these proteins between geriatric and adult brains. Parvalbumin- and calbindin-expressing neurons are relatively sensitive to degeneration in the cerebellum of older dogs. Parvalbumin labelling was associated with dystrophic structures that are commonly associated with ageing. Copyright 2002 Elsevier Science Ltd.

  20. The calcium-binding protein S100P in normal and malignant human tissues

    Directory of Open Access Journals (Sweden)

    Pastorek Jaromir

    2008-02-01

    Full Text Available Abstract Background S100P is a Ca2+ binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions. Methods We examined S100P expression in normal human tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. S100P protein expression was also studied in a series of tumors, consisting of 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas using a novel anti-S100P monoclonal antibody. Results Among the normal tissues, the highest S100P mRNA levels were observed in the placenta and esophagus. Moderate signals were also detected in the stomach, duodenum, large intestine, prostate and leukocytes. At the protein level, the highest reactions for S100P were seen in the placenta and stomach. Immunostaining of tumor specimens showed that S100P protein is expressed in all the tumor categories included in the study, being most prevalent in gastric tumors. Conclusion Based on our observations, S100P is widely expressed in both normal and malignant tissues. The high expression in some tumors suggests that it may represent a potential target molecule for future diagnostic and therapeutic applications.

  1. Structural Insights into Membrane Targeting by the Flagellar Calcium-binding Protein (FCaBP) a Myristoylated and Palmitoylated Calcium Sensor in Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    J Wingard; J Ladner; M Vanarotti; A Fisher; H Robinson; K Buchanan; D Engman; J Ames

    2011-12-31

    The flagellar calcium-binding protein (FCaBP) of the protozoan Trypanosoma cruzi is targeted to the flagellar membrane where it regulates flagellar function and assembly. As a first step toward understanding the Ca{sup 2+}-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca{sup 2+}-free state determined at 2.2{angstrom} resolution. The first 17 residues from the N terminus appear unstructured and solvent-exposed. Residues implicated in membrane targeting (Lys-19, Lys-22, and Lys-25) are flanked by an exposed N-terminal helix (residues 26-37), forming a patch of positive charge on the protein surface that may interact electrostatically with flagellar membrane targets. The four EF-hands in FCaBP each adopt a 'closed conformation' similar to that seen in Ca{sup 2+}-free calmodulin. The overall fold of FCaBP is closest to that of grancalcin and other members of the penta EF-hand superfamily. Unlike the dimeric penta EF-hand proteins, FCaBP lacks a fifth EF-hand and is monomeric. The unstructured N-terminal region of FCaBP suggests that its covalently attached myristoyl group at the N terminus may be solvent-exposed, in contrast to the highly sequestered myristoyl group seen in recoverin and GCAP1. NMR analysis demonstrates that the myristoyl group attached to FCaBP is indeed solvent-exposed in both the Ca{sup 2+}-free and Ca{sup 2+}-bound states, and myristoylation has no effect on protein structure and folding stability. We propose that exposed acyl groups at the N terminus may anchor FCaBP to the flagellar membrane and that Ca{sup 2+}-induced conformational changes may control its binding to membrane-bound protein targets..

  2. Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+

    Directory of Open Access Journals (Sweden)

    Kumar Shivesh

    2012-08-01

    Full Text Available Abstract Background EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins. Results In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 from Entamoeba histolytica (EhCaBP1. The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the 1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are both enthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinate with water in this structure. For all the metals, binding to the 3rd site is only entropy driven. Conclusion Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy. The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ bound structure, except for the lack of water coordination. Sr2+ coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the

  3. Molecular cloning of the apoptosis-related calcium-binding protein AsALG-2 in Avena sativa.

    Science.gov (United States)

    Hoat, Trinh Xuan; Nakayashiki, Hitoshi; Yang, Qian; Tosa, Yukio; Mayama, Shigeyuki

    2013-04-01

    Victorin, the host-selective toxin produced by the fungus Cochliobolus victoriae, induces programmed cell death (PCD) in victorin-sensitive oat lines with characteristic features of animal apoptosis, such as mitochondrial permeability transition, chromatin condensation, nuclear DNA laddering and rRNA/mRNA degradation. In this study, we characterized a calcium-binding protein, namely AsALG-2, which might have a role in the victorin-induced PCD. AsALG-2 is homologous to the Apoptosis-Linked Gene ALG-2 identified in mammalian cells. Northern blot analysis revealed that the accumulation of AsALG-2 transcripts increased during victorin-induced PCD, but not during necrotic cell death. Salicylic acid, chitosan and chitin strongly activated the expression of general defence response genes, such as PR-10; however, neither induced cell death nor the accumulation of AsALG-2 mRNA. Pharmacological studies indicated that victorin-induced DNA laddering and AsALG-2 expression were regulated through similar pathways. The calcium channel blocker, nifedipine, moderately inhibited the accumulation of AsALG-2 mRNA during cell death. Trifluoperazine (calmodulin antagonist) and K252a (serine-threonine kinase inhibitor) reduced the victorin-induced phytoalexin accumulation, but did not prevent the victorin-induced DNA laddering or accumulation of AsALG-2 mRNA. Taken together, our investigations suggest that there is a calcium-mediated signalling pathway in animal and plant PCD in common. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  4. The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion

    NARCIS (Netherlands)

    Dessing, M.C.; Tammaro, A.; Pulskens, W.P.C.; Teske, G.J.; Butter, L.M.; Claessen, N.; Eijk, M. van; Poll, T. van der; Vogl, T.; Roth, J.; Florquin, S.; Leemans, J.C.

    2015-01-01

    Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury.

  5. Nitrite-cured color and phosphate-mediated water binding of pork muscle proteins as affected by calcium in the curing solution.

    Science.gov (United States)

    Zhao, Jing; Xiong, Youling L

    2012-07-01

    Calcium is a mineral naturally present in water and may be included into meat products during processing thereby influencing meat quality. Phosphates improve myofibril swelling and meat water-holding capacity (WHC) but can be sensitive to calcium precipitation. In this study, pork shoulder meat was used to investigate the impact of calcium at 0, 250, and 500 ppm and phosphate type [sodium pyrophosphate (PP), tripolyphosphate (TPP), and hexametaphopshate (HMP)] at 10 mM on nitrite-cured protein extract color at various pH levels (5.5, 6.0, and 6.5) and crude myofibril WHC at pH 6.0. Neither calcium nor phosphates present in the curing brines significantly affected the cured color. Increasing the pH tended to promote the formation of metmyoglobin instead of nitrosylmyoglobin. The ability of PP to enhance myofibril WHC was hampered (P water binding by myofibrils. The depressed muscle fiber swelling responding to added calcium as evidenced by phase contrast microscopy substantiated, to a certain extent, the deleterious effect of calcium, suggesting that hardness of curing water can significantly affect the quality of cured meat products. Although not affecting nitrite-cured color, calcium hampers the efficacy of phosphates to promote water binding by muscle proteins, underscoring the importance of water quality for brine-enhanced meat products. © 2012 Institute of Food Technologists®

  6. Neuroprotective Effect of Ginseng against Alteration of Calcium Binding Proteins Immunoreactivity in the Mice Hippocampus after Radiofrequency Exposure

    Directory of Open Access Journals (Sweden)

    Dhiraj Maskey

    2013-01-01

    Full Text Available Calcium binding proteins (CaBPs such as calbindin D28-k, parvalbumin, and calretinin are able to bind Ca2+ with high affinity. Changes in Ca2+ concentrations via CaBPs can disturb Ca2+ homeostasis. Brain damage can be induced by the prolonged electromagnetic field (EMF exposure with loss of interacellular Ca2+ balance. The present study investigated the radioprotective effect of ginseng in regard to CaBPs immunoreactivity (IR in the hippocampus through immunohistochemistry after one-month exposure at 1.6 SAR value by comparing sham control with exposed and ginseng-treated exposed groups separately. Loss of dendritic arborization was noted with the CaBPs in the Cornu Ammonis areas as well as a decrease of staining intensity of the granule cells in the dentate gyrus after exposure while no loss was observed in the ginseng-treated group. A significant difference in the relative mean density was noted between control and exposed groups but was nonsignificant in the ginseng-treated group. Decrease in CaBP IR with changes in the neuronal staining as observed in the exposed group would affect the hippocampal trisynaptic circuit by alteration of the Ca2+ concentration which could be prevented by ginseng. Hence, ginseng could contribute as a radioprotective agent against EMF exposure, contributing to the maintenance of Ca2+ homeostasis by preventing impairment of intracellular Ca2+ levels in the hippocampus.

  7. Gene expression and protein localisation of calcyclin, a calcium-binding protein of the S-100 family in fresh neuroblastomas.

    Science.gov (United States)

    Tonini, G P; Fabretti, G; Kuznicki, J; Massimo, L; Scaruffi, P; Brisigotti, M; Mazzocco, K

    1995-01-01

    Calcyclin gene, a Ca(2+)-binding protein with homology to S-100, has been found to be expressed at different levels in leukaemic cells and in other tumour cells. We recently reported the expression of the gene in human neuroblastoma (NB) cell lines, and suggested a possible role of calcyclin in cell differentiation. To extend our findings, we investigated the expression of the gene in NB cells induced to differentiate by retinoic acid (RA), using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Time-course experiments employing LA-N-5 cells showed that calcyclin mRNA appeared 2 h after RA treatment, long before the cells were blocked in the G1 cell-cycle phase and before the neurite-like structures outgrew from the cell bodies. This suggests the involvement of the gene in the early phase of cell differentiation. Furthermore, we investigated mRNA expression in a series of fresh neuroblastomas. NB tumours showed a heterogeneous pattern of calcyclin expression, although calcyclin seemed to be expressed more frequently in cases with a favourable Shimada histology. We also studied the expression of the protein in formalin fixed and paraffin embedded tissues, by using a specific anticalcyclin antibody. The protein was detected in stromal cells which characterise a more mature histological type, and in nerve sheaths, whereas neuroblasts were negative. The tissue that expressed calcyclin protein showed a Schwann-like differentiation and, unlike S-100 protein, calcyclin was expressed in the perineurium.

  8. Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

    Energy Technology Data Exchange (ETDEWEB)

    Gourinath, S., E-mail: sgourinath@mail.jnu.ac.in; Padhan, Narendra; Alam, Neelima; Bhattacharya, Alok [School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067 (India)

    2005-04-01

    Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca{sup 2+}-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 Å, β = 116.7°. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 Å, β = 92.8°. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 Å, β = 97.9°.

  9. Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord.

    Science.gov (United States)

    Zhang, Ming-Dong; Barde, Swapnali; Szodorai, Edit; Josephson, Anna; Mitsios, Nicholas; Watanabe, Masahiko; Attems, Johannes; Lubec, Gert; Kovács, Gábor G; Uhlén, Mathias; Mulder, Jan; Harkany, Tibor; Hökfelt, Tomas

    2016-09-01

    Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k(+) neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2(+) and/or calretinin(+). Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.

  10. Subdivisions of the auditory midbrain (n. mesencephalicus lateralis, pars dorsalis in zebra finches using calcium-binding protein immunocytochemistry.

    Directory of Open Access Journals (Sweden)

    Priscilla Logerot

    Full Text Available The midbrain nucleus mesencephalicus lateralis pars dorsalis (MLd is thought to be the avian homologue of the central nucleus of the mammalian inferior colliculus. As such, it is a major relay in the ascending auditory pathway of all birds and in songbirds mediates the auditory feedback necessary for the learning and maintenance of song. To clarify the organization of MLd, we applied three calcium binding protein antibodies to tissue sections from the brains of adult male and female zebra finches. The staining patterns resulting from the application of parvalbumin, calbindin and calretinin antibodies differed from each other and in different parts of the nucleus. Parvalbumin-like immunoreactivity was distributed throughout the whole nucleus, as defined by the totality of the terminations of brainstem auditory afferents; in other words parvalbumin-like immunoreactivity defines the boundaries of MLd. Staining patterns of parvalbumin, calbindin and calretinin defined two regions of MLd: inner (MLd.I and outer (MLd.O. MLd.O largely surrounds MLd.I and is distinct from the surrounding intercollicular nucleus. Unlike the case in some non-songbirds, however, the two MLd regions do not correspond to the terminal zones of the projections of the brainstem auditory nuclei angularis and laminaris, which have been found to overlap substantially throughout the nucleus in zebra finches.

  11. Assessment of the relationship between the molecular properties of calcium channel blockers and plasma protein binding data

    Directory of Open Access Journals (Sweden)

    Odović Jadranka V.

    2017-01-01

    Full Text Available In this study we investigated the relationship between the calcium channel blockers (CCBs, amlodipine, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, verapamil and diltiazem, and their calculated molecular descriptors: polar surface area (PSA, molecular weight (Mw, volume value (Vol, aqueous solubility data (logS, lipophilicity (logP, acidity (pKa values and plasma protein binding (PPB data, obtained from relevant literature. The relationships between the computed molecular properties of selected CCBs and their PPB data were investigated by simple linear regression analysis that revealed very low correlations (R2<0.35. When multiple linear regression (MLR analysis was applied to investigate reliable correlations between the CCBs’ calculated molecular descriptors and PPB data, the best correlations were found for the relationships between CCBs, and PPB data and lipophilicity, and with application of the molecular descriptor (Mw, Vol, or pKa data as additional independent variables (R2=0.623; R2=0.741; R2=0.657, respectively, with an acceptable probability value (P<0.05, confirming that lipophilicity, together with other molecular properties, are essential for the drugs’ PPB. We conclude that this could be considered as an additional in vitro approach for modeling CCBs. [Projekat Ministarstva nauke Republike Srbije, br. TR34031

  12. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    Directory of Open Access Journals (Sweden)

    Saray Santamaría-Hernando

    Full Text Available Proteins of the animal heme peroxidase (ANP superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20, where it was found to be involved in Ca(2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+ binding with a K(D of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821 is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of

  13. A novel biomarker associated with distress in humans: calcium-binding protein, spermatid-specific 1 (CABS1).

    Science.gov (United States)

    Ritz, Thomas; Rosenfield, David; St Laurent, Chris D; Trueba, Ana F; Werchan, Chelsey A; Vogel, Pia D; Auchus, Richard J; Reyes-Serratos, Eduardo; Befus, A Dean

    2017-06-01

    Calcium-binding protein spermatid-specific 1 (CABS1) is expressed in the human submandibular gland and has an anti-inflammatory motif similar to that in submandibular rat 1 in rats. Here, we investigate CABS1 in human saliva and its association with psychological and physiological distress and inflammation in humans. Volunteers participated across three studies: 1) weekly baseline measures; 2) a psychosocial speech and mental arithmetic stressor under evaluative threat; and 3) during academic exam stress. Salivary samples were analyzed for CABS1 and cortisol. Additional measures included questionnaires of perceived stress and negative affect; exhaled nitric oxide; respiration and cardiac activity; lung function; and salivary and nasal inflammatory markers. We identified a CABS1 immunoreactive band at 27 kDa in all participants and additional molecular mass forms in some participants. One week temporal stability of the 27-kDa band was satisfactory (test-retest reliability estimate = 0.62-0.86). Acute stress increased intensity of 18, 27, and 55 kDa bands; 27-kDa increases were associated with more negative affect and lower heart rate, sympathetic activity, respiration rate, and minute ventilation. In both acute and academic stress, changes in 27 kDa were positively associated with salivary cortisol. The 27-kDa band was also positively associated with VEGF and salivary leukotriene B4 levels. Participants with low molecular weight CABS1 bands showed reduced habitual stress and negative affect in response to acute stress. CABS1 is readily detected in human saliva and is associated with psychological and physiological indicators of stress. The role of CABS1 in inflammatory processes, stress, and stress resilience requires careful study. Copyright © 2017 the American Physiological Society.

  14. Deficiency in Calcium-Binding Protein S100A4 Impairs the Adjuvant Action of Cholera Toxin

    Directory of Open Access Journals (Sweden)

    Jia-Bin Sun

    2017-09-01

    Full Text Available The calcium-binding protein S100A4 has been described to promote pathological inflammation in experimental autoimmune and inflammatory disorders and in allergy and to contribute to antigen presentation and antibody response after parenteral immunization with an alum-adjuvanted antigen. In this study, we extend these findings by demonstrating that mice lacking S100A4 have a defective humoral and cellular immune response to mucosal (sublingual immunization with a model protein antigen [ovalbumin (OVA] given together with the strong mucosal adjuvant cholera toxin (CT, and that this impairment is due to defective adjuvant-stimulated antigen presentation by antigen-presenting cells. In comparison to wild-type (WT mice, mice genetically lacking S100A4 had reduced humoral and cellular immune responses after immunization with OVA plus CT, including a complete lack of detectable germinal center reaction. Further, when stimulated in vitro with OVA plus CT, S100A4−/− dendritic cells (DCs showed impaired responses in several CT-stimulated immune regulatory molecules including the co-stimulatory molecule CD86, inflammasome-associated caspase-1 and IL-1β. Coculture of OVA-specific OT-II T cells with S100A4−/− DCs that had been pulse incubated with OVA plus CT resulted in impaired OT-II T cell proliferation and reduced production of Th1, Th2, and Th17 cytokines compared to similar cocultures with WT DCs. In accordance with these findings, transfection of WT DCs with S100A4-targeting small interfering RNA (siRNA but not mock-siRNA resulted in significant reductions in the expression of caspase-1 and IL-1β as well as CD86 in response to CT. Importantly, also engraftment of WT DCs into S100A4−/− mice effectively restored the immune response to immunization in the recipients. In conclusion, our results demonstrate that deficiency in S100A4 has a strong impact on the development of both humoral and cellular immunity after mucosal immunization using CT

  15. Comparative distribution of relaxin-3 inputs and calcium-binding protein-positive neurons in rat amygdala

    Directory of Open Access Journals (Sweden)

    Fabio N Santos

    2016-04-01

    Full Text Available The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or ‘hubs’ within these key circuits. One such input arises from the nucleus incertus (NI in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin, calretinin and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST and in the endopiriform

  16. The lack of relationships between vitamin D3 metabolites and calcium-binding protein in the eggshell gland of laying birds.

    Science.gov (United States)

    Bar, A; Rosenberg, J; Hurwitz, S

    1984-01-01

    The relationship of the metabolism of vitamin D3 and calcium-binding protein (CaBP) to calcium transport by the eggshell gland (ESG) was assessed in chickens. Plasma or ESG 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and ESG CaBP were no different between periods of ESG inactivity and of shell calcification. A severe dietary calcium deficiency resulted in increased kidney 25-hydroxycholecalciferol-1-hydroxylase activity (542%), plasma and ESG 1,25(OH)2D3 concentrations (193 and 274%, respectively), but in decreased ESG CaBP (34%), associated with the production of poorly calcified eggs. Significant correlations were found between 25 hydroxycholecalciferol-1-hydroxylase, plasma 1,25(OH)2D3 and ESG 1,25(OH)2D3, but not between ESG 1,25(OH)2D3 and CaBP. Hens with a low shell density had a significantly lower (55%) ESG CaBP than those with high shell density, without any significant change in ESG 1,25(OH)2D3. Significant correlations were found between ESG CaBP and shell calcium. Total receptors for 1,25(OH)2D3 were lower in ESG than in the intestine. The results suggest that CaBP level and calcium transport in the ESG are not regulated by 1,25(OH)2D3.

  17. Binding of calcium and carbonate to polyacrylates.

    Science.gov (United States)

    Tribello, Gareth A; Liew, CheeChin; Parrinello, Michele

    2009-05-21

    Polyacrylate molecules can be used to slow the growth of calcium carbonate. However, little is known about the mechanism by which the molecules impede the growth rate. A recent computational study (Bulo et al. Macromolecules 2007, 40, 3437) used metadynamics to investigate the binding of calcium to polyacrylate chains and has thrown some light on the coiling and precipitation of these polymers. We extend these simulations to examine the binding of calcium and carbonate to polyacrylate chains. We show that calcium complexed with both carbonate and polyacrylate is a very stable species. The free energies of calcium-carbonate-polyacrylate complexes, with different polymer configurations, are calculated, and differences in the free energy of the binding of carbonate are shown to be due to differences in the amount of steric hindrance about the calcium, which prevents the approach of the carbonate ion.

  18. The serum concentration of the calcium binding protein S100B is positively associated with cognitive performance in older adults

    Directory of Open Access Journals (Sweden)

    Virginie eLam

    2013-09-01

    Full Text Available S100B is a calcium-binding peptide produced predominantly by astroglial cells in the central nervous system. S100B paradoxically has neurotrophic and apoptotic effects, dependent on extracellular concentration. This study investigated the relationship between serum S100B levels and neuropsychological performance across a range of cognitive domains in healthy older aged adults. A cohort of 219 participants between the ages of 43 and 84 years (141 female were recruited. Subjects provided a fasting blood sample for S100B measurement (Mean = 0.24 ng/mL, SD = 0.14 and completed a battery of neuropsychological tests. S100B concentrations (both with and without the covariates of age and sex were positively associated with the following measures of cognitive performance: digit symbol coding, Stroop test and measures of verbal ability. The results from this study show that serum S100B is positively associated with better cognitive performance in healthy older adults.

  19. Comparative Analysis of Calcium-Binding Myeloid-Related Protein-8/14 in Saliva and Serum of Patients With Periodontitis and Healthy Individuals.

    Science.gov (United States)

    Haririan, Hady; Andrukhov, Oleh; Pablik, Eleonore; Neuhofer, Michaela; Moritz, Andreas; Rausch-Fan, Xiaohui

    2016-02-01

    This study aims to investigate calcium-binding myeloid-related protein (MRP)-8/14 in the saliva and serum of individuals with periodontitis and periodontally healthy individuals for the assessment of its role in the pathogenesis and clinical diagnosis of periodontitis. This cross-sectional study includes 56 patients with periodontitis and 44 periodontally healthy individuals. Saliva and serum were collected for the detection of MRP-8/14 and calcium levels. Periodontopathic bacteria were determined by polymerase chain reaction in saliva. Correlations between salivary and serum MRP-8/14 levels and clinical parameters, bacteria, and calcium were analyzed with Pearson correlation in a multiple regression model. MRP-8/14 levels were documented with receiver operating characteristic (ROC) curves. Compared with healthy individuals, MRP-8/14 levels were significantly higher in both the saliva and serum of patients with periodontitis, but calcium was increased only in saliva. A high diagnostic potential of salivary MRP-8/14 was detected for periodontitis (ROC = 0.86). Salivary MRP-8/14 levels correlated significantly with the presence of the periodontopathogen Treponema denticola, as well as with the clinical parameters of periodontitis. MRP-8/14 in saliva might be a potential diagnostic parameter for periodontal disease.

  20. Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

    2017-09-01

    Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

  1. The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus.

    Science.gov (United States)

    Bürstenbinder, Katharina; Möller, Birgit; Plötner, Romina; Stamm, Gina; Hause, Gerd; Mitra, Dipannita; Abel, Steffen

    2017-03-01

    Calcium (Ca 2+ ) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related calmodulin-like (CML) polypeptides are principal sensors of Ca 2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67 DOMAIN (IQD) family emerged as possibly the largest class of CaM-interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis ( Arabidopsis thaliana ) differentially localize, using green fluorescent protein (GFP)-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane subdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca 2+ -CaM signaling modules to specific subcellular sites for precise regulation of Ca 2+ -dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in Nicotiana benthamiana cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca 2+ signaling at multiple cellular sites to regulate cell function, shape, and growth. © 2017 American Society of Plant Biologists. All Rights Reserved.

  2. The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus1[OPEN

    Science.gov (United States)

    Plötner, Romina; Stamm, Gina; Hause, Gerd; Mitra, Dipannita; Abel, Steffen

    2017-01-01

    Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related calmodulin-like (CML) polypeptides are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67 DOMAIN (IQD) family emerged as possibly the largest class of CaM-interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis (Arabidopsis thaliana) differentially localize, using green fluorescent protein (GFP)-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane subdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in Nicotiana benthamiana cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth. PMID:28115582

  3. Changes of calcium binding proteins, c-Fos and COX in hippocampal formation and cerebellum of Niemann-Pick, type C mouse.

    Science.gov (United States)

    Byun, Kyunghee; Kim, Daesik; Bayarsaikhan, Enkhjaigal; Oh, Jeehyun; Kim, Jisun; Kwak, Grace; Jeong, Goo-Bo; Jo, Seung-Mook; Lee, Bonghee

    2013-09-01

    Niemann-Pick disease, type C (NPC) is an intractable disease that is accompanied by ataxia, dystonia, neurodegeneration, and dementia due to an NPC gene defect. Disruption of calcium homeostasis in neurons is important in patients with NPC. Thus, we used immunohistochemistry to assess the expression levels of calcium binding proteins (calbindin D28K, parvalbumin, and calretinin), c-Fos and cyclooxygenase-1,2 (COX-1,2) in the hippocampal formation and cerebellum of 4 and 8 week old NPC+/+, NPC+/-, and NPC-/- mice. General expression of these proteins decreased in the hippocampus and cerebellum of NPC-/- compared to that in both young and adult NPC+/+ or NPC+/- mice. Parvalbumin, COX-1,2 or c-Fos-immunoreactive neurons were widely detected in the CA1, CA3, and DG of the hippocampus, but the immunoreactivities were decreased sharply in all areas of hippocampus of NPC-/- compared to NPC+/+ and NPC+/- mice. Taken together, reduction of these proteins may be one of the strong phenotypes related to the neuronal degeneration in NPC-/- mice. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Acute Sleep Deprivation Increases Serum Levels of Neuron-Specific Enolase (NSE) and S100 Calcium Binding Protein B (S-100B) in Healthy Young Men

    Science.gov (United States)

    Benedict, Christian; Cedernaes, Jonathan; Giedraitis, Vilmantas; Nilsson, Emil K.; Hogenkamp, Pleunie S.; Vågesjö, Evelina; Massena, Sara; Pettersson, Ulrika; Christoffersson, Gustaf; Phillipson, Mia; Broman, Jan-Erik; Lannfelt, Lars; Zetterberg, Henrik; Schiöth, Helgi B.

    2014-01-01

    Study Objectives: To investigate whether total sleep deprivation (TSD) affects circulating concentrations of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in humans. These factors are usually found in the cytoplasm of neurons and glia cells. Increasing concentrations of these factors in blood may be therefore indicative for either neuronal damage, impaired blood brain barrier function, or both. In addition, amyloid β (Aβ) peptides 1-42 and 1-40 were measured in plasma to calculate their ratio. A reduced plasma ratio of Aβ peptides 1-42 to 1-40 is considered an indirect measure of increased deposition of Aβ 1-42 peptide in the brain. Design: Subjects participated in two conditions (including either 8-h of nocturnal sleep [22:30-06:30] or TSD). Fasting blood samples were drawn before and after sleep interventions (19:30 and 07:30, respectively). Setting: Sleep laboratory. Participants: 15 healthy young men. Results: TSD increased morning serum levels of NSE (P = 0.002) and S-100B (P = 0.02) by approximately 20%, compared with values obtained after a night of sleep. In contrast, the ratio of Aβ peptides 1-42 to 1-40 did not differ between the sleep interventions. Conclusions: Future studies in which both serum and cerebrospinal fluid are sampled after sleep loss should elucidate whether the increase in serum neuron-specific enolase and S100 calcium binding protein B is primarily caused by neuronal damage, impaired blood brain barrier function, or is just a consequence of increased gene expression in non-neuronal cells, such as leukocytes. Citation: Benedict C; Cedernaes J; Giedraitis V; Nilsson EK; Hogenkamp PS; Vågesjö E; Massena S; Pettersson U; Christoffersson G; Phillipson M; Broman JE; Lannfelt L; Zetterberg H; Schiöth HB. Acute Sleep Deprivation Increases Serum Levels of Neuron-Specific Enolase (NSE) and S100 Calcium Binding Protein B (S-100B) in Healthy Young Men. SLEEP 2014;37(1):195-198. PMID:24470708

  5. Enhanced expression of a calcium-dependent protein kinase from ...

    Indian Academy of Sciences (India)

    Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

  6. Calcium binding to low molecular weight compounds and health promoting products

    DEFF Research Database (Denmark)

    Vavrusova, Martina

    absorption. Therefore, calcium as an essential nutrient should not be underestimated in our diet. Milk and dairy products are good sources of bioavailable calcium due to specific protein binding. Other sources of calcium, apart from a balanced and healthy diet, are calcium supplements and calcium fortified......Calcium precipitation in the almost neutral environment of the intestines is a process related to weight loss management and plays an important role in the prevention of colon cancer development. This process also affects calcium bioavailability which is decreased due to decreased calcium...... food. Therefore, an understanding of the basic chemistry of calcium binding to low molecular weight compounds can contribute to a general knowledge about calcium bioavailability and also to product improvement. Calcium precipitation with palmitate was described by a first-order reaction for conditions...

  7. [Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei].

    Science.gov (United States)

    Wang, Cai-xia; Huang, Jian-fang; Xiang, Jun-jian; Sun, Yi-fan; Lv, Si; Guo, Jie

    2012-08-01

    To identify sarcoplasmic calcium-binding protein (SCP) as a minor shrimp allergen by mass spectrometry, and to analyze the immune cross-reactivity among crustacean SCPs. The M(r); 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS. BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans. The puritifed M(r); 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting. The M(r); 21 000 shrimp allergen was identified as SCP. Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans. Western blotting showed that the proteins with M(r); about 21 000, corresponding to SCP from Metapenaeus ensis, Penaeus monodon, Oratosquilla oratoria, Macrobrachium rosenbergii, Procambarus clarkii, Portunus pelagicus, Charybdis feriatus, Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei. SCP is a minor shrimp allergen, and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans, which can be used as detective, diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.

  8. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  9. Prenatal acoustic stimulation influences neuronal size and the expression of calcium-binding proteins (calbindin D-28K and parvalbumin) in chick hippocampus.

    Science.gov (United States)

    Chaudhury, Sraboni; Nag, Tapas Chandra; Wadhwa, Shashi

    2006-12-01

    Prenatal auditory enrichment by species-specific sounds and sitar music enhances the expression of immediate early genes, synaptic proteins and calcium binding proteins (CaBPs) as well as modifies the structural components of the brainstem auditory nuclei and auditory imprinting area in chicks. There is also facilitation of postnatal auditory preference of the chicks to maternal calls following both types of sound stimulation indicating prenatal perceptual learning. To examine whether the sound enrichment protocol also affects the areas related to learning and memory, we assessed morphological changes in the hippocampus at post-hatch day 1 of control and prenatally sound-stimulated chicks. Additionally, the proportions of neurons containing calbindin D-28K and parvalbumin immunoreactivity as well as their protein levels were determined. Fertilized eggs of domestic chick were incubated under normal conditions of temperature, humidity, forced draft of air as well as light and dark (12:12h) photoperiods. They were exposed to patterned sounds of species-specific and sitar music at 65 dB for 15 min per hour over a day/night cycle from day 10 of incubation till hatching. The hippocampal volume, neuronal nuclear size and total number of neurons showed a significant increase in the music-stimulated group as compared to the species-specific sound-stimulated and control groups. However, in both the auditory-stimulated groups the protein levels of calbindin and parvalbumin as well as the percentage of the immunopositive neurons were increased. The enhanced proportion of CaBPs in the sound-enriched groups suggests greater Ca(2+) influx, which may influence long-term potentiation and short-term memory.

  10. A calcium-binding protein, rice annexin OsANN1, enhances heat stress tolerance by modulating the production of H2O2.

    Science.gov (United States)

    Qiao, Bei; Zhang, Qian; Liu, Dongliang; Wang, Haiqi; Yin, Jingya; Wang, Rui; He, Mengli; Cui, Meng; Shang, Zhonglin; Wang, Dekai; Zhu, Zhengge

    2015-09-01

    OsANN1 is a member of the annexin protein family in rice. The function of this protein and the mechanisms of its involvement in stress responses and stress tolerance are largely unknown. Here it is reported that OsANN1 confers abiotic stress tolerance by modulating antioxidant accumulation under abiotic stress. OsANN1-knockdown [RNA interference (RNAi)] plants were more sensitive to heat and drought stresses, whereas OsANN1-overexpression (OE) lines showed improved growth with higher expression of OsANN1 under abiotic stress. Overexpression of OsANN1 promoted SOD (superoxide dismutase) and CAT (catalase) activities, which regulate H2O2 content and redox homeostasis, suggesting the existence of a feedback mechanism between OsANN1 and H2O2 production under abiotic stress. Higher expression of OsANN1 can provide overall cellular protection against abiotic stress-induced damage, and a significant accumulation of OsANN1-green fluorescent protein (GFP) signals was found in the cytosol after heat shock treatment. OsANN1 also has calcium-binding and ATPase activities in vitro, indicating that OsANN1 has multiple functions in rice growth. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that OsANN1 interacts with OsCDPK24. This cross-talk may provide additional layers of regulation in the abiotic stress response. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Calcium ion-protein interactions in prothrombin activation

    Energy Technology Data Exchange (ETDEWEB)

    Brenckle, G.M.; Carlisle, T.L.; Jackson, C.M.

    1981-01-01

    1. The protein concentration dependence observed in the calcium binding to fragment 1 indicates that calcium-mediated dimerization is responsible for the cooperative calcium binding behavior usually observed. ''Unusual'' fragment 1, which exhibits negative cooperativity (the type of binding behavior expected for ions interacting with a charged protein) at high concentration, also exhibit altered self-association behavior. 2. The calcium-induced spectral perturbations that are observed by fluorescence and ultraviolet difference spectroscopy are influenced by calcium-mediated dimerization. Similar spectral perturbations may also be induced by other divalent, trivalent, and monovalent ions, as well as changes in pH. Because this is a multi-site system, only limited interpretation of the spectral data is possible without calcium binding data. 3. Although strong side chain CD signals make estimation of fragment 1 secondary structure ambiguous, the CD data do indicate small changes in structure during calcium binding. Similar changes are observed upon addition of monovalent ions at high concentration or after lowering the pH. No coupling between changes in conformation and the cooperative calcium binding behavior has yet been observed to exist.

  12. Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

    Directory of Open Access Journals (Sweden)

    Sukhdeep Kumar

    Full Text Available Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD varieties. We establish (a that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT, resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

  13. Protein kinase C interaction with calcium: a phospholipid-dependent process.

    LENUS (Irish Health Repository)

    Bazzi, M D

    1990-08-21

    The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

  14. Expression of calcium-binding proteins and selected neuropeptides in the human, chimpanzee, and crab-eating macaque claustrum.

    Science.gov (United States)

    Pirone, Andrea; Castagna, Maura; Granato, Alberto; Peruffo, Antonella; Quilici, Francesca; Cavicchioli, Laura; Piano, Ilaria; Lenzi, Carla; Cozzi, Bruno

    2014-01-01

    The claustrum is present in all mammalian species examined so far and its morphology, chemoarchitecture, physiology, phylogenesis and ontogenesis are still a matter of debate. Several morphologically distinct types of immunostained cells were described in different mammalian species. To date, a comparative study on the neurochemical organization of the human and non-human primates claustrum has not been fully described yet, partially due to technical reasons linked to the postmortem sampling interval. The present study analyze the localization and morphology of neurons expressing parvalbumin (PV), calretinin (CR), NPY, and somatostatin (SOM) in the claustrum of man (# 5), chimpanzee (# 1) and crab-eating monkey (# 3). Immunoreactivity for the used markers was observed in neuronal cell bodies and processes distributed throughout the anterior-posterior extent of human, chimpanzee and macaque claustrum. Both CR- and PV-immunoreactive (ir) neurons were mostly localized in the central and ventral region of the claustrum of the three species while SOM- and NPY-ir neurons seemed to be equally distributed throughout the ventral-dorsal extent. In the chimpanzee claustrum SOM-ir elements were not observed. No co-localization of PV with CR was found, thus suggesting the existence of two non-overlapping populations of PV and CR-ir interneurons. The expression of most proteins (CR, PV, NPY), was similar in all species. The only exception was the absence of SOM-ir elements in the claustrum of the chimpanzee, likely due to species specific variability. Our data suggest a possible common structural organization shared with the adjacent insular region, a further element that emphasizes a possible common ontogeny of the claustrum and the neocortex.

  15. Expression of calcium-binding proteins and selected neuropeptides in the human, chimpanzee, and crab-eating macaque claustrum

    Directory of Open Access Journals (Sweden)

    Andrea ePirone

    2014-05-01

    Full Text Available The claustrum is present in all mammalian species examined so far and its morphology, chemoarchitecture, physiology, phylogenesis and ontogenesis are still a matter of debate. Several morphologically distinct types of immunostained cells were described in different mammalian species. To date, a comparative study on the neurochemical organization of the human and non-human primates claustrum has not been fully described yet, partially due to technical reasons linked to the postmortem sampling interval. The present study analyzes the localization and morphology of neurons expressing parvalbumin (PV, calretinin (CR, NPY, and somatostatin (SOM in the claustrum of man (# 5, chimpanzee (# 1 and crab-eating monkey (#3. Immunoreactivity for the used markers was observed in neuronal cell bodies and processes distributed throughout the anterior-posterior extent of human, chimpanzee and macaque claustrum. Both CR- and PV-immunoreactive (ir neurons were mostly localized in the central and ventral region of the claustrum of the three species while SOM- and NPY-ir neurons seemed to be equally distributed throughout the ventral-dorsal extent. In the chimpanzee claustrum SOM-ir elements were not observed. No co-localization of PV with CR was found, thus suggesting the existence of two non-overlapping populations of PV and CR-ir interneurons. The expression of most proteins (CR, PV, NPY, was similar in all species. The only exception was the absence of SOM-ir elements in the claustrum of the chimpanzee, likely due to species specific variability. Our data suggest a possible common structural organization shared with the adjacent insular region, a further element that emphasizes a possible common ontogeny of the claustrum and the neocortex.

  16. Intracellular amyloid-β accumulation in calcium-binding protein-deficient neurons leads to amyloid-β plaque formation in animal model of Alzheimer's disease.

    Science.gov (United States)

    Moon, Minho; Hong, Hyun-Seok; Nam, Dong Woo; Baik, Sung Hoon; Song, Hyundong; Kook, Sun-Young; Kim, Yong Soo; Lee, Jeewoo; Mook-Jung, Inhee

    2012-01-01

    One of the major hallmarks of Alzheimer's disease (AD) is the extracellular deposition of amyloid-β (Aβ) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aβ deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aβ (iAβ) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aβ deposits had several shapes and sizes, and that iAβ occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aβ (N3pE-Aβ), which has been suggested to be a seeding molecule for senile plaques, inside the Aβ plaques only after iAβ accumulation, which argues against its seeding role. In addition, we found that iAβ accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aβ-independent accumulation of Aβ in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aβ plaques are formed, what happens to iAβ and how Aβ causes selective neuronal loss in AD patients.

  17. Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

    DEFF Research Database (Denmark)

    Nielbo, Steen; Thomsen, Jens K; Graversen, Jonas Heilskov

    2004-01-01

    Tetranectin is a homotrimeric protein containing a C-type lectin-like domain. This domain (TN3) can bind calcium, but in the absence of calcium, the domain binds a number of kringle-type protein ligands. Two of the calcium-coordinating residues are also critical for binding plasminogen kringle 4 (K...

  18. Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

    Science.gov (United States)

    Lu, Defen; Shang, Guijun; Zhang, Heqiao; Yu, Qian; Cong, Xiaoyan; Yuan, Jupeng; He, Fengjuan; Zhu, Chunyuan; Zhao, Yanyu; Yin, Kun; Chen, Yuanyuan; Hu, Junqiang; Zhang, Xiaodan; Yuan, Zenglin; Xu, Sujuan; Hu, Wei; Cang, Huaixing; Gu, Lichuan

    2014-06-01

    The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex. © 2014 John Wiley & Sons Ltd.

  19. Analytical models of calcium binding in a calcium channel

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jinn-Liang [Department of Applied Mathematics, National Hsinchu University of Education, Hsinchu 300, Taiwan (China); Eisenberg, Bob [Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois 60612 (United States)

    2014-08-21

    The anomalous mole fraction effect of L-type calcium channels is analyzed using a Fermi like distribution with the experimental data of Almers and McCleskey [J. Physiol. 353, 585 (1984)] and the atomic resolution model of Lipkind and Fozzard [Biochemistry 40, 6786 (2001)] of the selectivity filter of the channel. Much of the analysis is algebraic, independent of differential equations. The Fermi distribution is derived from the configuration entropy of ions and water molecules with different sizes, different valences, and interstitial voids between particles. It allows us to calculate potentials and distances (between the binding ion and the oxygen ions of the glutamate side chains) directly from the experimental data using algebraic formulas. The spatial resolution of these results is comparable with those of molecular models, but of course the accuracy is no better than that implied by the experimental data. The glutamate side chains in our model are flexible enough to accommodate different types of binding ions in different bath conditions. The binding curves of Na{sup +} and Ca{sup 2+} for [CaCl{sub 2}] ranging from 10{sup −8} to 10{sup −2} M with a fixed 32 mM background [NaCl] are shown to agree with published Monte Carlo simulations. The Poisson-Fermi differential equation—that includes both steric and correlation effects—is then used to obtain the spatial profiles of energy, concentration, and dielectric coefficient from the solvent region to the filter. The energy profiles of ions are shown to depend sensitively on the steric energy that is not taken into account in the classical rate theory. We improve the rate theory by introducing a steric energy that lumps the effects of excluded volumes of all ions and water molecules and empty spaces between particles created by Lennard-Jones type and electrostatic forces. We show that the energy landscape varies significantly with bath concentrations. The energy landscape is not constant.

  20. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Lindblom, Karin

    2009-01-01

    The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin......, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment...... LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen...

  1. S100 calcium binding protein B as a biomarker of delirium duration in the intensive care unit – an exploratory analysis

    Directory of Open Access Journals (Sweden)

    Khan BA

    2013-12-01

    Full Text Available Babar A Khan,1–3 Mark O Farber,1 Noll Campbell,2–5 Anthony Perkins,2,3 Nagendra K Prasad,6 Siu L Hui,1–3 Douglas K Miller,1–3 Enrique Calvo-Ayala,1 John D Buckley,1 Ruxandra Ionescu,1 Anantha Shekhar,1 E Wesley Ely,7,8 Malaz A Boustani1–3 1Indiana University School of Medicine, 2Indiana University Center for Aging Research, 3Regenstrief Institute, Inc., 4Wishard Health Services, Indianapolis, 5Department of Pharmacy Practice, Purdue University College of Pharmacy, West Lafayette, 6Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, 7Vanderbilt University School of Medicine, 8VA Tennessee Valley Geriatric Research Education Clinical Center (GRECC, Nashville, TN, USA Background: Currently, there are no valid and reliable biomarkers to identify delirious patients predisposed to longer delirium duration. We investigated the hypothesis that elevated S100 calcium binding protein B (S100β levels will be associated with longer delirium duration in critically ill patients. Methods: A prospective observational cohort study was performed in the medical, surgical, and progressive intensive care units (ICUs of a tertiary care, university affiliated, and urban hospital. Sixty-three delirious patients were selected for the analysis, with two samples of S100β collected on days 1 and 8 of enrollment. The main outcome measure was delirium duration. Using the cutoff of <0.1 ng/mL and $0.1 ng/mL as normal and abnormal levels of S100β, respectively, on day 1 and day 8, four exposure groups were created: Group A, normal S100β levels on day 1 and day 8; Group B, normal S100β level on day 1 and abnormal S100β level on day 8; Group C, abnormal S100β level on day 1 and normal on day 8; and Group D, abnormal S100β levels on both day 1 and day 8. Results: Patients with abnormal levels of S100β showed a trend towards higher delirium duration (P=0.076; Group B (standard deviation (7.0 [3.2] days, Group C (5.5 [6.3] days, and Group D

  2. Kinetics of calcium binding to dental biofilm bacteria.

    Science.gov (United States)

    Leitão, Tarcísio Jorge; Cury, Jaime Aparecido; Tenuta, Livia Maria Andaló

    2018-01-01

    Dental biofilm bacteria can bind calcium ions and release them during a pH drop, which could decrease the driving force for dental demineralization (i.e. hydroxyapatite dissolution) occurring at reduced pHs. However, the kinetics of this binding and release is not completely understood. Here we validated a method to evaluate the kinetics of calcium binding and release to/from Streptococcus mutans, and estimated the importance of this reservoir as a source of ions. The kinetics of calcium binding was assessed by measuring the amount of bound calcium in S. mutans Ingbrit 1600 pellets treated with PIPES buffer, pH 7.0, containing 1 or 10 mM Ca; for the release kinetics, bacterial pellets previously treated with 1 mM or 10 mM Ca were exposed to the calcium-free or 1 mM Ca PIPES buffer, pH 7.0, for up to 60 min. Binding and release curves were constructed and parameters of kinetics were calculated. Also, calcium release was assessed by exposing pellets previously treated with calcium to a pH 5.0 buffer for 10 min. Calcium binding to bacteria was concentration-dependent and rapid, with maximum binding reached at 5 min. On the other hand, calcium release was slower, and according to the calculations, would never be complete in the groups pretreated with 10 mM Ca. Decreasing pH from 7.0 to 5.0 caused a release of calcium able to increase the surrounding fluid calcium concentration in 2 mM. The results suggest that dental biofilm bacteria may act as a calcium reservoir, rapidly binding ions from surrounding fluids, releasing them slowly at neutral pH and promptly during a pH drop.

  3. Calcium Binding Ability of Recombinant Buffalo Regucalcin: A Study Using Circular Dichroism Spectroscopy.

    Science.gov (United States)

    Harikrishna, P; Thomas, Jobin; Shende, A M; Bhure, S K

    2017-04-01

    Regucalcin is a calcium regulating multifunctional protein reported to have many important functions like calcium homeostasis, anti-oxidative, anti-apoptotic and anti-cancerous functions. Although it is demonstrated as a calcium regulating protein, the calcium binding ability of regucalcin is still a controversy. The main reason for the controversy is that it lacks a typical EF hand motif which is common to most of the calcium binding proteins. Even though many studies reported regucalcin as a calcium binding protein, there are some studies reporting regucalcin as non-calcium binding also. In the present study, we investigated the calcium binding ability of recombinant buffalo regucalcin by assessing the secondary structural changes of the protein using circular dichroism spectroscopy after adding Ca(2+) to the protein solution. Two types of calcium binding studies were done, one with different concentration of calcium chloride (0.5 mM CaCl2, 1 mM CaCl2, 2 mM CaCl2) and other at different time interval (no incubation and 10 min incubation) after addition of calcium chloride. Significant structural changes were observed in both studies which prove the calcium binding ability of recombinant regucalcin. A constant increase in the α-helix (1.1% with 0.5 mM CaCl2, 1.4% with 1 mM CaCl2, 3.5% with 2 mM CaCl2) and a decrease in β-sheets (78.5% with 0.5 mM CaCl2, 77.4% with 1 mM CaCl2, 75.7% with 2 mM CaCl2) were observed with the increase in calcium chloride concentration. There was a rapid increase in α-helix and decrease in β-sheets immediately after addition of calcium chloride, which subsides after 10 min incubation.

  4. Interaction entropy for protein-protein binding

    Science.gov (United States)

    Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

    2017-03-01

    Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

  5. Calcium Carbonate Formation by Genetically Engineered Inorganic Binding Peptides

    Science.gov (United States)

    Gresswell, Carolyn Gayle

    Understanding how organisms are capable of forming (synthesize, crystallize, and organize) solid minerals into complex architectures has been a fundamental question of biomimetic materials chemistry and biomineralization for decades. This study utilizes short peptides selected using a cell surface display library for the specific polymorphs of calcium carbonate, i.e., aragonite and calcite, to identify two sets of sequences which can then be used to examine their effects in the formation, crystal structure, morphology of the CaCO3 minerals. A procedure of counter selection, along with fluorescence microscopy (FM) characterization, was adapted to insure that the sequences on the cells were specific to their respective substrate, i.e., aragonite or calcite. From the resulting two sets of sequences selected, five distinct strong binders were identified with a variety of biochemical characteristics and synthesized for further study. Protein derived peptides, using the known sequences of the proteins that are associated with calcite or aragonite, were also designed using a bioinformatics-based similarity analysis of the two sets of binders. In particular, an aragonite binding protein segment, AP7, a protein found in nacre, was chosen for this design and the resulting effects of the designed peptides and the AP7 were examined. Specifically, the binding affinities of the selected and the protein derived peptides off the cells were then tested using FM; these studies resulted in different binding characteristics of the synthesized and cellular bound peptides. Two of the peptides that displayed strong binding on the cells bound to neither of the CaCO 3 substrates and both the high and low similarity protein-derived peptides bound to both polymorphs. However, two of the peptides were found to only bind to their respective polymorph showing; these results are significant in that with this study it is demonstrated that the designed peptides based on experimental library

  6. Protein-Mediated Precipitation of Calcium Carbonate.

    Science.gov (United States)

    Polowczyk, Izabela; Bastrzyk, Anna; Fiedot, Marta

    2016-11-22

    Calcium carbonate is an important component in exoskeletons of many organisms. The synthesis of calcium carbonate was performed by mixing dimethyl carbonate and an aqueous solution of calcium chloride dihydrate. The precipitation product was characterized by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) measurements. In addition, the turbidity of the reaction solution was acquired to monitor the kinetics of the calcium carbonate structure's growth in the investigated system. In this study, samples of CaCO₃ particles obtained with individual proteins, such as ovalbumin, lysozyme, and a mixture of the proteins, were characterized and compared with a control sample, i.e., synthesized without proteins. The obtained data indicated that the addition of ovalbumin to the reaction changed the morphology of crystals from rhombohedral to 'stack-like' structures. Lysozyme, however, did not affect the morphology of calcium carbonate, yet the presence of the protein mixture led to the creation of more complex composites in which the calcium carbonate crystals were constructed in protein matrices formed by the ovalbumin-lysozyme interaction. It was also observed that in the protein mixture, ovalbumin has a major influence on the CaCO₃ formation through a strong interaction with calcium ions, which leads to the coalescence and creation of a steric barrier reducing particle growth. The authors proposed a mechanism of calcium carbonate grain growth in the presence of both proteins, taking into account the interaction of calcium ions with the protein.

  7. Calcium binding and homoassociation of E-cadherin domains.

    Science.gov (United States)

    Koch, A W; Pokutta, S; Lustig, A; Engel, J

    1997-06-24

    Cadherins are single pass transmembrane glycoproteins which mediate calcium dependent cell-cell adhesion by homophilic interactions. To reveal the molecular details of calcium binding and homoassociation, we recombinantly expressed in Escherichia coli a domain pair consisting of the first two domains of E-cadherin (ECAD12) and the single domains 1, 2, and 5. ECAD12 encompasses the most N-terminal of the four putative calcium-binding pockets in the extracellular region of E-cadherin. Equilibrium dialysis experiments revealed that the single domains do not bind Ca2+, but ECAD12 was found to bind three calcium ions. ECAD12 dimerizes (Kd = 0.08 +/- 0.02 mM) in the presence of Ca2+ as we could demonstrate by analytical ultracentrifugation. Calcium binding to ECAD12 induces conformational changes which were monitored by electrophoretic mobility and by circular dichroism. By analyzing our equilibrium dialysis data with a single binding site model, we found an average Kd of 460 microM for the three bound Ca2+. Assuming a model for three binding sites, which slightly increased the quality of the fit, we obtained two identical Kds of 330 microM and a third much higher Kd of 2 mM. The entire extracellular region of E-cadherin, which was recombinantly expressed in mammalian cells, binds nine Ca2+ with a much lower average Kd of 30 microM. Therefore, we conclude that the four calcium binding pockets are not identical. Since binding to ECAD12 occurs at Ca2+ concentrations close to those in the extracellular space, we suggest that the N-terminal domain pair might be involved in calcium regulation of E-cadherin mediated cell-cell adhesion.

  8. Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation.

    Science.gov (United States)

    Sanagavarapu, Kalyani; Weiffert, Tanja; Ní Mhurchú, Niamh; O'Connell, David; Linse, Sara

    2016-01-01

    Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation.

  9. Blood Levels of S-100 Calcium-Binding Protein B, High-Sensitivity C-Reactive Protein, and Interleukin-6 for Changes in Depressive Symptom Severity after Coronary Artery Bypass Grafting: Prospective Cohort Nested within a Randomized, Controlled Trial

    Science.gov (United States)

    Pearlman, Daniel M.; Brown, Jeremiah R.; MacKenzie, Todd A.; Hernandez, Felix; Najjar, Souhel

    2014-01-01

    Background Cross-sectional and retrospective studies have associated major depressive disorder with glial activation and injury as well as blood–brain barrier disruption, but these associations have not been assessed prospectively. Here, we aimed to determine the relationship between changes in depressive symptom severity and in blood levels of S-100 calcium-binding protein B (S-100B), high-sensitivity C-reactive protein, and interleukin-6 following an inflammatory challenge. Methods Fifty unselected participants were recruited from a randomized, controlled trial comparing coronary artery bypass grafting procedures performed with versus without cardiopulmonary bypass for the risk of neurocognitive decline. Depressive symptom severity was measured at baseline, discharge, and six-month follow-up using the Beck Depression Inventory II (BDI-II). The primary outcome of the present biomarker study was acute change in depressive symptom severity, defined as the intra-subject difference between baseline and discharge BDI-II scores. Blood biomarker levels were determined at baseline and 2 days postoperative. Results Changes in S-100B levels correlated positively with acute changes in depressive symptom severity (Spearman ρ, 0.62; P = 0.0004) and accounted for about one-fourth of their observed variance (R2, 0.23; P = 0.0105). This association remained statistically significant after adjusting for baseline S-100B levels, age, weight, body-mass index, or β-blocker use, but not baseline BDI-II scores (P = 0.064). There was no statistically significant association between the primary outcome and baseline S-100B levels, baseline high-sensitivity C-reactive protein or interleukin-6 levels, or changes in high-sensitivity C-reactive protein or interleukin-6 levels. Among most participants, levels of all three biomarkers were normal at baseline and markedly elevated at 2 days postoperative. Conclusions Acute changes in depressive symptom severity were specifically

  10. Protein-Mediated Precipitation of Calcium Carbonate

    Directory of Open Access Journals (Sweden)

    Izabela Polowczyk

    2016-11-01

    Full Text Available Calcium carbonate is an important component in exoskeletons of many organisms. The synthesis of calcium carbonate was performed by mixing dimethyl carbonate and an aqueous solution of calcium chloride dihydrate. The precipitation product was characterized by means of scanning electron microscopy (SEM, transmission electron microscopy (TEM, X-ray diffraction (XRD, and Fourier transform infrared spectroscopy (FTIR measurements. In addition, the turbidity of the reaction solution was acquired to monitor the kinetics of the calcium carbonate structure’s growth in the investigated system. In this study, samples of CaCO3 particles obtained with individual proteins, such as ovalbumin, lysozyme, and a mixture of the proteins, were characterized and compared with a control sample, i.e., synthesized without proteins. The obtained data indicated that the addition of ovalbumin to the reaction changed the morphology of crystals from rhombohedral to ‘stack-like’ structures. Lysozyme, however, did not affect the morphology of calcium carbonate, yet the presence of the protein mixture led to the creation of more complex composites in which the calcium carbonate crystals were constructed in protein matrices formed by the ovalbumin-lysozyme interaction. It was also observed that in the protein mixture, ovalbumin has a major influence on the CaCO3 formation through a strong interaction with calcium ions, which leads to the coalescence and creation of a steric barrier reducing particle growth. The authors proposed a mechanism of calcium carbonate grain growth in the presence of both proteins, taking into account the interaction of calcium ions with the protein.

  11. Protein-Mediated Precipitation of Calcium Carbonate

    Science.gov (United States)

    Polowczyk, Izabela; Bastrzyk, Anna; Fiedot, Marta

    2016-01-01

    Calcium carbonate is an important component in exoskeletons of many organisms. The synthesis of calcium carbonate was performed by mixing dimethyl carbonate and an aqueous solution of calcium chloride dihydrate. The precipitation product was characterized by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) measurements. In addition, the turbidity of the reaction solution was acquired to monitor the kinetics of the calcium carbonate structure’s growth in the investigated system. In this study, samples of CaCO3 particles obtained with individual proteins, such as ovalbumin, lysozyme, and a mixture of the proteins, were characterized and compared with a control sample, i.e., synthesized without proteins. The obtained data indicated that the addition of ovalbumin to the reaction changed the morphology of crystals from rhombohedral to ‘stack-like’ structures. Lysozyme, however, did not affect the morphology of calcium carbonate, yet the presence of the protein mixture led to the creation of more complex composites in which the calcium carbonate crystals were constructed in protein matrices formed by the ovalbumin-lysozyme interaction. It was also observed that in the protein mixture, ovalbumin has a major influence on the CaCO3 formation through a strong interaction with calcium ions, which leads to the coalescence and creation of a steric barrier reducing particle growth. The authors proposed a mechanism of calcium carbonate grain growth in the presence of both proteins, taking into account the interaction of calcium ions with the protein. PMID:28774065

  12. Calcium binding to the purple membrane : A molecular dynamics study

    NARCIS (Netherlands)

    Wassenaar, Tsjerk A.; Daura, Xavier; Padros, Esteve; Mark, Alan E.

    2009-01-01

    The purple membrane (PM) is a specialized membrane patch found in halophilic archaea, containing the photoreceptor bacteriorhodopsin (bR). It is long known that calcium ions bind to the PM, but their position and role remain elusive to date. Molecular dynamics simulations in conjunction with a

  13. Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

    Science.gov (United States)

    Wyatt, Sarah E.; Tsou, Pei-Lan; Robertson, Dominique; Brown, C. S. (Principal Investigator)

    2002-01-01

    Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

  14. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  15. Viral infection controlled by a calcium-dependent lipid-binding module in ALIX.

    Science.gov (United States)

    Bissig, Christin; Lenoir, Marc; Velluz, Marie-Claire; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael; Gruenberg, Jean

    2013-05-28

    ALIX plays a role in nucleocapsid release during viral infection, as does lysobisphosphatidic acid (LBPA). However, the mechanism remains unclear. Here we report that LBPA is recognized within an exposed site in ALIX Bro1 domain predicted by MODA, an algorithm for discovering membrane-docking areas in proteins. LBPA interactions revealed a strict requirement for a structural calcium tightly bound near the lipid interaction site. Unlike other calcium- and phospholipid-binding proteins, the all-helical triangle-shaped fold of the Bro1 domain confers selectivity for LBPA via a pair of hydrophobic residues in a flexible loop, which undergoes a conformational change upon membrane association. Both LBPA and calcium binding are necessary for endosome association and virus infection, as are ALIX ESCRT binding and dimerization capacity. We conclude that LBPA recruits ALIX onto late endosomes via the calcium-bound Bro1 domain, triggering a conformational change in ALIX to mediate the delivery of viral nucleocapsids to the cytosol during infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding.

    Science.gov (United States)

    Pokutta, S; Herrenknecht, K; Kemler, R; Engel, J

    1994-08-01

    The cell-adhesion protein E-cadherin/uvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147-154]. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca2+, the circular dichroic spectra indicated predominantly beta-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average K(d) values of 45-150 microM. Cleavage of the protein fragment by trypsin occurred only at low Ca2+ concentrations and from the calcium-dependence of cleavage rates, a K(d) value of 24 microM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.

  17. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  18. Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors.

    Directory of Open Access Journals (Sweden)

    Konstantin Denessiouk

    Full Text Available Proteins with EF-hand calcium-binding motifs are essential for many cellular processes, but are also associated with cancer, autism, cardiac arrhythmias, and Alzheimer's, skeletal muscle and neuronal diseases. Functionally, all EF-hand proteins are divided into two groups: (1 calcium sensors, which function to translate the signal to various responses; and (2 calcium buffers, which control the level of free Ca2+ ions in the cytoplasm. The borderline between the two groups is not clear, and many proteins cannot be described as definitive buffers or sensors. Here, we describe two highly-conserved structural motifs found in all known different families of the EF-hand proteins. The two motifs provide a supporting scaffold for the DxDxDG calcium binding loop and contribute to the hydrophobic core of the EF hand domain. The motifs allow more precise identification of calcium buffers and calcium sensors. Based on the characteristics of the two motifs, we could classify individual EF-hand domains into five groups: (1 Open static; (2 Closed static; (3 Local dynamic; (4 Dynamic; and (5 Local static EF-hand domains.

  19. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Bokhovchuk, F.M.; Vuister, G.W.

    2012-01-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal

  20. Oxygen-binding haem proteins.

    Science.gov (United States)

    Wilson, Michael T; Reeder, Brandon J

    2008-01-01

    Myoglobin and haemoglobin, the respiratory pigments of mammals and some molluscs, annelids and arthropods, belong to an ancient superfamily of haem-associated globin proteins. Members of this family share common structural and spectral features. They also share some general functional characteristics, such as the ability to bind ligands, e.g. O2, CO and NO, at the iron atom and to undergo redox changes. These properties are used in vivo to perform a wide range of biochemical and physiological roles. While it is acknowledged that the major role of haemoglobin is to bind oxygen reversibly and deliver it to the tissues, this is not its only function, while the often-stated role of myoglobin as an oxygen storage protein is possibly a misconception. Furthermore, haemoglobin and myoglobin express enzymic activities that are important to their function, e.g. NO dioxygenase activity or peroxidatic activity that may be partly responsible for pathophysiology following haemorrhage. Evidence for these functions is described, and the discussion extended to include proteins that have recently been discovered and that are expressed at low levels within the cell. These proteins are hexaco-ordinate, unlike haemoglobin and myoglobin, and are widely distributed throughout the animal kingdom (e.g. neuroglobins and cytoglobins). They may have specialist roles in oxygen delivery to particular sites within the cell but may also perform roles associated with O2 sensing and signalling and in responses to stress, e.g. protection from reactive oxygen and nitrogen species. Haemoglobins are also widespread in plants and bacteria and may serve similar protective functions.

  1. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  2. Engineering RNA-binding proteins for biology.

    Science.gov (United States)

    Chen, Yu; Varani, Gabriele

    2013-08-01

    RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequence specificity will provide valuable tools for biochemical research as well as potential therapeutic applications. In this review, we discuss the suitability of various RNA-binding domains for engineering RNA-binding specificity, based on the structural basis for their recognition. We also compare various protein engineering and design methods applied to RNA-binding proteins, and discuss future applications of these proteins. © 2013 FEBS.

  3. The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

    Science.gov (United States)

    Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

    Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

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

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    and semen. The function and significance of FBPs are unresolved, however, it has been suggested that they may facilitate folate uptake, e.g. during suckling. The present study shows that megalin, a large, multiligand endocytic receptor and member of the low-density lipoprotein-receptor family, is able...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...

  5. Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Tiago J Dantas

    Full Text Available Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC, stabilising it, and its presence slightly increases nucleotide excision repair (NER activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

  6. Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes.

    Science.gov (United States)

    Ocampo, Marisol; Vera, Ricardo; Eduardo Rodriguez, Luis; Curtidor, Hernando; Urquiza, Mauricio; Suarez, Jorge; Garcia, Javier; Puentes, Alvaro; Lopez, Ramsés; Trujillo, Mary; Torres, Elizabeth; Patarroyo, Manuel Elkin

    2002-01-01

    Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.

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

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

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

  8. The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV.

    Science.gov (United States)

    Maurer, P; Hohenadl, C; Hohenester, E; Göhring, W; Timpl, R; Engel, J

    1995-10-20

    The extracellular glycoprotein BM-40 consists of three domains, an acidic domain I, a follistatin (FS)-like domain II and a calcium-binding EC domain with an EF-hand related motif. BM-40 and several other related proteins (QR1, SC1/hevin, testican and tsc-36/FRP) are members of a novel modular protein family that share the FS domain followed by an EC domain. We have expressed this pair of FS and EC domains (mutant delta I) and the calcium-binding EC domain alone (mutant delta I, II) of human BM-40 as recombinant proteins in human 293 cells. Circular dichroism demonstrated that both mutants were obtained as folded proteins with a distinct three-dimensional conformation. In addition, mutant delta I, II could be readily crystallized and diffraction patterns with a resolution limit of 2.4 A resolution were obtained. Calcium binding to this fragment was ten times weaker (Kd = 0.8 microM) than for the wild-type protein. Identical reversible increases in alpha-helicity upon calcium binding were observed for the 150-residue long mutant delta I, II and for BM-40 (286 residues). A 26-residue synthetic peptide corresponding to the EF-hand related motif exhibited much weaker calcium binding. The apparent dissociation constant decreased with increasing peptide concentration (from Kd 2.4 mM at 1 microM, to Kd 0.3 mM at 100 microM peptide concentration) and calcium binding was accompanied by dimerization of the peptide. This suggests that for strong calcium binding the EF-hand related motif has to be embedded into a larger protein domain that can form an autonomously folding protein module. The EC domain was also shown by surface plasmon resonance assay to be responsible for calcium-dependent binding to collagen IV with an affinity (Kd = 19 microM) only sixfold lower than that of intact human BM-40.

  9. Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium*

    Science.gov (United States)

    Zhang, Chen; Huang, Yun; Jiang, Yusheng; Mulpuri, Nagaraju; Wei, Ling; Hamelberg, Donald; Brown, Edward M.; Yang, Jenny J.

    2014-01-01

    Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and l-Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The heterocommunication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events. PMID:24394414

  10. binding protein gene from Dasypyrum Villosum

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... common wheat. Blue copper-binding protein (BCB) can bind a single copper atom (Ryden and Hunt, 1993). The copper binding sites consist of two ..... Moerschbacher BM, Noll U, Gorrichon L, Reisener HJ (1990). Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust.

  11. Testosterone increases urinary calcium excretion and inhibits expression of renal calcium transport proteins

    DEFF Research Database (Denmark)

    Hsu, Yu-Juei; Dimke, Henrik Anthony; Schoeber, Joost P H

    2010-01-01

    Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had...... higher urinary calcium excretion than female mice and their renal calcium transporters were expressed at a lower level. We also found that orchidectomized mice excreted less calcium in their urine than sham-operated control mice and that the hypocalciuria was normalized after testosterone replacement...... calcium transport. Thus, our study shows that gender differences in renal calcium handling are, in part, mediated by the inhibitory actions of androgens on TRPV5-mediated active renal calcium transport....

  12. [Calcium/protein relation of women on the climacteric].

    Science.gov (United States)

    Montilla, Regina das Neves Girão; Aldrighi, José Mendes; Marucci, Maria de Fátima Nunes

    2004-01-01

    To evaluate the calcium/protein relation of the diet of climacteric women. In a transversal study the diet was evaluated of 154 women 35-65 years old and matriculated in the Health Clinic of the Climateric Woman of Health Center of Public Health College of the São Paulo University. The food intake of calcium and protein was investigated by "24 hours recall" method. The evaluation of calcium/protein relation was made according to Massey and Heaney (1998), that is 20/1 (mg/g). The mean of intake of calcium was 624.9 mg, the mean of intake of protein was 86.7 g and the calcium/protein relation was 7/1 (624.9 mg/86.7 g). The studied population presents inadequated consumation of the calcium and protein nutrients. It could result in serious risk for health.

  13. The complexity of minocycline serum protein binding.

    Science.gov (United States)

    Zhou, Jian; Tran, Brian T; Tam, Vincent H

    2017-06-01

    Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines.

  14. Adenovirus DNA binding protein: helix destabilising properties.

    OpenAIRE

    Monaghan, A; Webster, A; Hay, R T

    1994-01-01

    Adenovirus DNA binding protein is a multifunctional protein essential for viral DNA replication. To investigate the role of the DNA binding protein in this process its interaction with partial DNA duplexes was examined. Duplex regions of DNA, created when a short DNA strand is annealed to its complementary sequence present in the single stranded form of M13 phage DNA, were efficiently unwound by DNA binding protein in a reaction that required neither ATP nor MgCl2. The unwinding activity of D...

  15. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  16. A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix*

    Science.gov (United States)

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-01-01

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBankTM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate. PMID:20150428

  17. Calcium binding and voltage gating in Cx46 hemichannels.

    Science.gov (United States)

    Pinto, Bernardo I; Pupo, Amaury; García, Isaac E; Mena-Ulecia, Karel; Martínez, Agustín D; Latorre, Ramón; Gonzalez, Carlos

    2017-11-20

    The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca 2+ ) and membrane voltage. Electrophysiological and atomic force microscopy experiments indicate that Ca 2+ stabilizes the hemichannel closed state. However, structural data show that Ca 2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca 2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca 2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca 2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca 2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca 2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca 2+ inhibits the water flux through Cx hemichannels. We conclude that Ca 2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.

  18. Functions of Intracellular Retinoid Binding-Proteins.

    Science.gov (United States)

    Napoli, Joseph L

    Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

  19. SVOP is a nucleotide binding protein.

    Directory of Open Access Journals (Sweden)

    Jia Yao

    Full Text Available BACKGROUND: Synaptic Vesicle Protein 2 (SV2 and SV2-related protein (SVOP are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1. In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties. METHODOLOGY/PRINCIPAL FINDINGS: We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[gamma] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9-12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A. CONCLUSIONS/SIGNIFICANCE: SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately.

  20. Calcium ion binding to a soil fulvic acid using a Donnan Potential model

    Energy Technology Data Exchange (ETDEWEB)

    Marinsky, J.A. [State Univ. of New York, Albany, NY (United States). Dept. of Chemistry; Mathuthu, A. [National Univ. of Science and Technology, Bulawayo (Zimbabwe). Dept. of Applied Chemistry; Ephraim, J.H. [Linkoeping Univ. (Sweden). Dept. of Theme Research, Water and Environmental Studies; Reddy, M.M. [Geological Survey, Boulder, CO (United States)

    1999-10-01

    Calcium ion binding to a soil fulvic acid (Armadale Bh Horizon) was evaluated over a range of calcium ion concentrations, from pH 3.8 to 7.3, using potentiometric titrations and calcium ion electrode measurements. Fulvic acid concentration was constant (100 milligrams per liter) and calcium ion concentration varied up to 8 x 10{sup -4} moles per liter. Experiments discussed here included: (1) titrations of fulvic acid-calcium ion containing solutions with sodium hydroxide; and (2) titrations of fully neutralized fulvic acid with calcium chloride solutions. Apparent binding constants (expressed as the logarithm of the value, log {beta}{sub app}) vary with solution pH, calcium ion concentration, degree of acid dissociation, and ionic strength (from log {beta}{sub app}=2.5 to 3.9) and are similar to those reported by others. Fulvic acid charge, and the associated Donnan Potential, influences calcium ion-fulvic acid ion pair formation. A Donnan Potential correction term allowed calculation of intrinsic calcium ion-fulvic acid binding constants. Intrinsic binding constants vary from 1.2 to 2.5 (the average value is about log {beta}=1.6) and are similar to, but somewhat higher than, stability constants for calcium ion-carboxylic acid monodentate complexes. (orig.)

  1. Crystallographic Analysis of Calcium-dependent Heparin Binding to Annexin A2

    Energy Technology Data Exchange (ETDEWEB)

    Shao,C.; Zhang, F.; Kemp, M.; Lindhardt, R.; Waisman, D.; Head, J.; Seaton, B.

    2006-01-01

    Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

  2. CAP binding proteins associated with the nucleus.

    Science.gov (United States)

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in initiation factors as well as in preparations of crude nuclei. It was released from nuclei by washing with buffer of moderate salt concentration. Three high molecular weight cap binding proteins (approximately 120 000, approximately 89 000, approximately 80 000 dalton) were found in the nuclear fraction and were only partly released upon nuclease digestion and high salt extraction. Images PMID:6889136

  3. Calcium binding to low molecular weight compounds and health promoting products

    DEFF Research Database (Denmark)

    Vavrusova, Martina

    to acidic amino acids and dipeptides was also investigated. It was found that aspartate binds calcium stronger than glutamate. This was confirmed by electrochemical determination of the association constants for aqueous solutions at ionic strength 0.20 and 1.0. The mixed dipeptides Asp/Glu showed additive...... affinity of calcium binding as determined by the association constants and also compared with the values predicted from the individual amino acids. In contrast, the AspAsp dipeptide showed an affinity less than additive, while the GluGlu dipeptide showed affinity of calcium binding to be more than additive....... Additionally, the affinity of calcium to serine was small but was increased for the SerGlu dipeptide and the values became comparable to phosphorylated serine. An increase in ionic strength lead to decreased calcium binding of the studied free amino acids, dipeptides and phosphorylated serine as expected...

  4. Binding pocket optimization by computational protein design.

    Directory of Open Access Journals (Sweden)

    Christoph Malisi

    Full Text Available Engineering specific interactions between proteins and small molecules is extremely useful for biological studies, as these interactions are essential for molecular recognition. Furthermore, many biotechnological applications are made possible by such an engineering approach, ranging from biosensors to the design of custom enzyme catalysts. Here, we present a novel method for the computational design of protein-small ligand binding named PocketOptimizer. The program can be used to modify protein binding pocket residues to improve or establish binding of a small molecule. It is a modular pipeline based on a number of customizable molecular modeling tools to predict mutations that alter the affinity of a target protein to its ligand. At its heart it uses a receptor-ligand scoring function to estimate the binding free energy between protein and ligand. We compiled a benchmark set that we used to systematically assess the performance of our method. It consists of proteins for which mutational variants with different binding affinities for their ligands and experimentally determined structures exist. Within this test set PocketOptimizer correctly predicts the mutant with the higher affinity in about 69% of the cases. A detailed analysis of the results reveals that the strengths of PocketOptimizer lie in the correct introduction of stabilizing hydrogen bonds to the ligand, as well as in the improved geometric complemetarity between ligand and binding pocket. Apart from the novel method for binding pocket design we also introduce a much needed benchmark data set for the comparison of affinities of mutant binding pockets, and that we use to asses programs for in silico design of ligand binding.

  5. Expressão da proteína ligadora de cálcio S100 A7 (psoriasina no carcinoma laríngeo Expression of calcium binding protein S100 A7 (psoriasin in laryngeal carcinoma

    Directory of Open Access Journals (Sweden)

    Rogério Costa Tiveron

    2012-08-01

    Full Text Available Muitos estudos relatam o aumento da expressão de S100 A7 (psoriasina em lesões neoplásicas. Destacam-se trabalhos em carcinoma da mama, espinocelular da bexiga, pele e cavidade oral. Não foi demonstrada expressão da S100 A7 em câncer de laringe. OBJETIVO: Identificar a expressão da proteína ligadora de cálcio S100 A7 e sua correlação com carcinomas espinocelular da laringe. MATERIAL E MÉTODOS: Amostras de tecido neoplásico de 63 pacientes foram submetidos à imunohis toquímica com o anticorpo S110 A7. Os resultados foram classificados e comparados. RESULTADOS: O grupo bem diferenciado teve a maior pontuação de falha no tratamento. O grupo moderadamente diferenciado apresentou escores mais elevados do que o grupo pouco diferenciado. Pontuações mais altas predominaram nos estágios I e II no grupo moderadamente diferenciado, enquanto a distribuição do escore foi mais homogênea em estados avançados (III e IV. Em relação às falhas no tratamento, o grupo pontuação zero (04/03 complicações: 75% diferiu significativamente da pontuação restante (13/59: 22%. CONCLUSÕES: A S100 A7 foi expressa em 93,7% dos casos de câncer de laringe, com maior positividade nos tumores mais diferenciados e taxa significativamente menor de falha no tratamento. A pontuação obtida não teve impacto sobre a sobrevivência.Many studies have reported increased expression of S100 A7 (psoriasin in neoplastic lesions. Among them are studies on breast carcinoma, bladder squamous cell carcinoma, skin tumors and oral cavity squamous cell carcinoma. The expression of S100 A7 has not been described for laryngeal cancer. OBJECTIVE: This study aims to identify the expression of the calcium-binding protein S100 A7 and its correlation with squamous cell carcinomas of the larynx. MATERIAL AND METHODS: Specimens from 63 patients were submitted to immunohistochemistry testing with antibody S100 A7. Results were classified and compared. RESULTS: The group with

  6. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    Science.gov (United States)

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p sperm were treated with milk followed by seminal plasma, coating of sperm with BSP proteins was not significantly reduced (57.6 %; p > 0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  7. Telomere-binding proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Zentgraf, U

    1995-02-01

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

  8. Lipid binding proteins from parasitic platyhelminthes

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2012-01-01

    Two main families of lipid binding proteins have been identified in parasitic Platyhelminthes: hydrophobic ligand binding proteins (HLBPs) and fatty acid binding proteins (FABPs). Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesize their own lipids, these lipid-binding proteins are important molecules in these organisms. HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates. Despite that the knowledge of their function is scarce, the differences in their molecular organization, ligand preferences, intra/extracellular localization, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment. PMID:22988444

  9. Lipid Binding Proteins from Parasitic Platyhelmithes

    Directory of Open Access Journals (Sweden)

    Gabriela eAlvite

    2012-09-01

    Full Text Available Two main families of lipid binding proteins have been identified in parasitic Platyhelminthes: hydrophobic ligand binding proteins (HLBPs and fatty acid binding proteins (FABPs. Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesise their own lipids, these lipid-binding proteins are important molecules in these organisms.HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates.Despite that the knowledge of their function is scarce, the differences in their molecular organisation, ligand preferences, intra/extracellular localisation, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment.

  10. Protein-Mediated Precipitation of Calcium Carbonate

    OpenAIRE

    Izabela Polowczyk; Anna Bastrzyk; Marta Fiedot

    2016-01-01

    Calcium carbonate is an important component in exoskeletons of many organisms. The synthesis of calcium carbonate was performed by mixing dimethyl carbonate and an aqueous solution of calcium chloride dihydrate. The precipitation product was characterized by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) measurements. In addition, the turbidity of the reaction solution was acquire...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate...... defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP...

  12. Haptenation: Chemical Reactivity and Protein Binding

    Directory of Open Access Journals (Sweden)

    Itai Chipinda

    2011-01-01

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

  13. Binding interactions of niclosamide with serum proteins

    Directory of Open Access Journals (Sweden)

    Esra Maltas

    2014-12-01

    Full Text Available A study of the binding of niclosamide (NC to serum proteins such as human serum albumin, hemoglobin, and globulin was carried out using fluorescence and UV-visible spectroscopy. Interactions between NC and these proteins were estimated by Stern–Volmer and van't Hoff equations. The binding constants and the thermodynamic parameters, ΔH, ΔS, and ΔG at different temperatures were also determined by using these equations. Data showed that NC may exhibit a static quenching mechanism with all proteins. The thermodynamic parameters were calculated. Data showed that van der Waals interactions and hydrogen bonds are the main forces for human serum albumin and hemoglobin. Globulin, however, bound to NC via hydrophobic interaction. The spectral changes of synchronous fluorescence suggested that both the microenvironment of NC and the conformation of the proteins changed in relation to their concentrations during NC's binding.

  14. The effects of calcium regulation of endosperm reserve protein ...

    African Journals Online (AJOL)

    The effects of steep liquor calcium ion on sorghum endosperm reserve protein mobilization were evaluated using two improved Nigeria sorghum cultivars (ICSV 400 and KSV 8). The key protein modification factors evaluated were free amino nitrogen (FAN), total non protein nitrogen (TNPN) and soluble protein of cold water ...

  15. Protein (Cyanobacteria): 495464111 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available emolysin-type calcium-binding repeat protein Moorea producens MATIQGNNGDNSLIGTANNDMMWGYDGNDTLWGQDGNDQLIGGNGSDQLIGGFGNDTLWGEDGNDT...LWGENGNDQLMGGSGNDTLWGEDGNDTLWGENGNDQLIGGNGSDQLIGGSGNDTLWGEDGNDTLWGGSGNDTLQGQDGNDQLIGDGGF

  16. Protein (Cyanobacteria): 230294 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available calcium-binding repeat protein Moorea producens 3L MATIQGNNGDNSLIGTANNDMMWGYDGNDTLWGQDGNDQLIGGNGSDQLIGGFGNDTLWGEDGNDT...LWGENGNDQLMGGSGNDTLWGEDGNDTLWGENGNDQLIGGNGSDQLIGGSGNDTLWGEDGNDTLWGGSGNDTLQGQDGNDQLIGDGGFNDLIGGSGADQFVLSTEGFTTITDFEDGIDRIQVSNRTFNDLGFILSGLPNSLIIVDNITGFGIGSLLNVSQGDITAADFIA ...

  17. Computational search for aflatoxin binding proteins

    Science.gov (United States)

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

    2017-10-01

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

  18. Improved mapping of protein binding sites

    Science.gov (United States)

    Kortvelyesi, Tamas; Silberstein, Michael; Dennis, Sheldon; Vajda, Sandor

    2003-02-01

    Computational mapping methods place molecular probes - small molecules or functional groups - on a protein surface in order to identify the most favorable binding positions by calculating an interaction potential. Mapping is an important step in a number of flexible docking and drug design algorithms. We have developed improved algorithms for mapping protein surfaces using small organic molecules as molecular probes. The calculations reproduce the binding of eight organic solvents to lysozyme as observed by NMR, as well as the binding of four solvents to thermolysin, in good agreement with x-ray data. Application to protein tyrosine phosphatase 1B shows that the information provided by the mapping can be very useful for drug design. We also studied why the organic solvents bind in the active site of proteins, in spite of the availability of alternative pockets that can very tightly accommodate some of the probes. A possible explanation is that the binding in the relatively large active site retains a number of rotational states, and hence leads to smaller entropy loss than the binding elsewhere else. Indeed, the mapping reveals that the clusters of the ligand molecules in the protein's active site contain different rotational-translational conformers, which represent different local minima of the free energy surface. In order to study the transitions between different conformers, reaction path and molecular dynamics calculations were performed. Results show that most of the rotational states are separated by low free energy barriers at the experimental temperature, and hence the entropy of binding in the active site is expected to be high.

  19. Binding characteristics of galectin-3 fusion proteins.

    Science.gov (United States)

    Böcker, Sophia; Elling, Lothar

    2017-05-01

    Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy. © The Author 2017. Published by Oxford

  20. Randomized crossover study comparing the phosphate-binding efficacy of calcium ketoglutarate versus calcium carbonate in patients on chronic hemodialysis

    DEFF Research Database (Denmark)

    Bro, S; Rasmussen, R A; Handberg, J

    1998-01-01

    The objective of the study was to evaluate the phosphate-binding efficacy, side effects, and cost of therapy of calcium ketoglutarate granulate as compared with calcium carbonate tablets in patients on chronic hemodialysis. The study design used was a randomized, crossover open trial, and the main...... outcome measurements were plasma ionized calcium levels, plasma phosphate levels, plasma intact parathyroid hormone (PTH) levels, requirements for supplemental aluminum-aminoacetate therapy, patient tolerance, and cost of therapy. Nineteen patients on chronic hemodialysis were treated with a dialysate......, diarrhea, general uneasiness), whereas the remaining 12 patients did not experience any side effects at all. The five patients with calcium ketoglutarate intolerance all had pre-existing gastrointestinal symptoms; four of them had received treatment with cimetidine or omeprazol before inclusion...

  1. Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

    Science.gov (United States)

    Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

    2016-09-01

    Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  2. The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats

    Directory of Open Access Journals (Sweden)

    Xu Yu-Dong

    2010-08-01

    Full Text Available Abstract Background The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR, which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma. Methods In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism. Results In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses. Conclusions Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.

  3. AUXIN BINDING PROTEIN1: The Outsider

    Science.gov (United States)

    Sauer, Michael; Kleine-Vehn, Jürgen

    2011-01-01

    AUXIN BINDING PROTEIN1 (ABP1) is one of the first characterized proteins that bind auxin and has been implied as a receptor for a number of auxin responses. Early studies characterized its auxin binding properties and focused on rapid electrophysiological and cell expansion responses, while subsequent work indicated a role in cell cycle and cell division control. Very recently, ABP1 has been ascribed a role in modulating endocytic events at the plasma membrane and RHO OF PLANTS-mediated cytoskeletal rearrangements during asymmetric cell expansion. The exact molecular function of ABP1 is still unresolved, but its main activity apparently lies in influencing events at the plasma membrane. This review aims to connect the novel findings with the more classical literature on ABP1 and to point out the many open questions that still separate us from a comprehensive model of ABP1 action, almost 40 years after the first reports of its existence. PMID:21719690

  4. Effect of calcium-binding peptide from Pacific cod (Gadus macrocephalus) bone on calcium bioavailability in rats.

    Science.gov (United States)

    Peng, Zhe; Hou, Hu; Zhang, Kai; Li, Bafang

    2017-04-15

    Bone collagen peptide with high affinity to Ca was extracted from Pacific cod (Gadus macrocephalus) bone. FTIR spectra of calcium-binding bone collagen peptide showed that band at 3381cm(-1) shifted to 3361cm(-1), 1455cm(-1) moved to 1411cm(-1), and amide II became deeper valley, compared with that of bone collagen peptide. This peptide was sequenced by Q-TOF-MS and sequences of Gly-Pro-Glu-Gly, Gly-Glu-Lys, Gly-Pro-Leu-Gly and Gly-Leu-Pro-Gly appeared repeatedly in some peptides. From SEM, after chelated with calcium, the loose and porous structure turned into granular structure. From the animal experiment, Ca apparent absorption rate, Ca retention rate and femur Ca content of calcium-binding bone collagen peptide group were significantly higher than those of model and CaCO3 groups (P<0.05), while serum ALP was significantly lower than model group (P<0.05) and similar to control group. The results suggested that calcium-binding bone collagen peptide could improve bioavailability of Ca and thus prevented Ca deficiency. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. /sup 3/H)Nitrendipine binding to calcium channels in bovine and rat pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Titeler, M.; De Souza, E.B.; Kuhar, M.J.

    1985-06-01

    (/sup 3/H)Nitrendipine was used to label sites in homogenates of bovine anterior and neurointermediate lobes of the pituitary gland. The amount of specific binding in the anterior lobe was 1.82 +/- 0.30 pmol/g wet weight of tissue and the KD was 1.44 +/- 0.02 X 10(-10) M. Preliminary experiments indicated a similar amount of binding in bovine neurointermediate lobe. In competition studies nimodipine and nisoldipine (two potent voltage-sensitive calcium channel blockers) displayed IC50 values of 1.6 and 6.8 X 10(-10) M, respectively. Verapamil and the verapamil-like calcium channel blockers D-600 and tiapamil competed in a complex manner for the (/sup 3/H)nitrendipine specific binding to bovine anterior pituitary homogenates. Autoradiographical studies demonstrated specific (/sup 3/H)nitrendipine binding sites distributed approximately equally in the anterior and posterior lobes, but not in the intermediate lobe of the rat pituitary. In general the properties of (/sup 3/H)nitrendipine binding in the pituitary tissue resemble strongly the properties of (/sup 3/H)nitrendipine binding in the brain which is believed to be to voltage-sensitive calcium channels. These results provide support for the hypothesis that calcium channels are involved in pituitary hormone secretion and that drugs that interact with calcium channels may modulate the secretory process directly at the level of the pituitary.

  6. Protein-Protein Interactions: Structurally Conserved Residues Distinguish between Binding Sites and Exposed Protein Surfaces

    National Research Council Canada - National Science Library

    Buyong Ma; Tal Elkayam; Haim Wolfson; Ruth Nussinov

    2003-01-01

    Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface...

  7. Quantifying drug-protein binding in vivo.

    Energy Technology Data Exchange (ETDEWEB)

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-02-17

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS.

  8. Diversity in protein profiles of individual calcium oxalate kidney stones.

    Science.gov (United States)

    Okumura, Nobuaki; Tsujihata, Masao; Momohara, Chikahiro; Yoshioka, Iwao; Suto, Kouzou; Nonomura, Norio; Okuyama, Akihiko; Takao, Toshifumi

    2013-01-01

    Calcium oxalate kidney stones contain low amounts of proteins, some of which have been implicated in progression or prevention of kidney stone formation. To gain insights into the pathophysiology of urolithiasis, we have characterized protein components of calcium oxalate kidney stones by proteomic approaches. Proteins extracted from kidney stones showed highly heterogeneous migration patterns in gel electrophoresis as reported. This was likely to be mainly due to proteolytic degradation and protein-protein crosslinking of Tamm-Horsfall protein and prothrombin. Protein profiles of calcium oxalate kidney stones were obtained by in-solution protease digestion followed by nanoLC-MALDI-tandem mass spectrometry, which resulted in identification of a total of 92 proteins in stones from 9 urolithiasis patients. Further analysis showed that protein species and their relative amounts were highly variable among individual stones. Although proteins such as prothrombin, osteopontin, calgranulin A and calgranulin B were found in most stones tested, some samples had high contents of prothrombin and osteopontin, while others had high contents of calgranulins. In addition, calgranulin-rich stones had various neutrophil-enriched proteins such as myeloperoxidase and lactotransferrin. These proteomic profiles of individual kidney stones suggest that multiple systems composed of different groups of proteins including leucocyte-derived ones are differently involved in pathogenesis of individual kidney stones depending on situations.

  9. End binding proteins are obligatory dimers.

    Directory of Open Access Journals (Sweden)

    Indrani Sen

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

  10. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

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

    2010-01-01

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

  11. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism.

    Science.gov (United States)

    Georgiou, Dimitra K; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M; Wang, Hui; Lagor, William R; Pautler, Robia G; Dirksen, Robert T; Hamilton, Susan L

    2015-09-25

    Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Fragment molecular orbital method for studying lanthanide interactions with proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tsushima, Satoru [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Komeiji, Y. [National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba (Japan); Mochizuki, Y. [Rikkyo Univ., Tokyo (Japan)

    2017-06-01

    The binding affinity of the calcium-binding protein calmodulin towards Eu{sup 3+} was studied as a model for lanthanide protein interactions in the large family of ''EF-hand'' calcium-binding proteins.

  13. Conformational changes produced by ATP binding to the plasma membrane calcium pump

    OpenAIRE

    Mangialavori, Irene C.; Ferreira Gomes, Mariela S.; Saffioti, Nicolas A.; Gonzalez-Lebrero, Rodolfo Martin; Rossi, Rolando Carlos; Rossi, Juan Pablo Francisco

    2017-01-01

    The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholin...

  14. The Medicago truncatula DMI1 protein modulates cytosolic calcium signaling

    DEFF Research Database (Denmark)

    Peiter, Edgar; Sun, Jongho; Heckmann, Anne Birgitte Lau

    2007-01-01

    nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities...... to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K+) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts...... as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium...

  15. Alcohol binding to the odorant binding protein LUSH: multiple factors affecting binding affinities.

    Science.gov (United States)

    Ader, Lauren; Jones, David N M; Lin, Hai

    2010-07-27

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH-alcohol complexes identify a set of specific hydrogen-bonding interactions as critical for optimal binding of ethanol. A set of truncated models based on the structure of the LUSH-butanol complex were constructed for the wild-type and mutant (T57S, S52A, and T57A) proteins in complexes with a series of n-alcohols and for the apoprotein bound to water and for the ligand-free protein. Using both gas-phase calculations and continuum solvation model calculations, we found that the widely used DFT model, B3LYP, failed to reproduce the experimentally observed trend of increasing binding affinity with the increasing length of the alkyl chain in the alcohol. In contrast, the recently developed M05-2X DFT model successfully reproduced this subtle trend. Analysis of the results indicated that multiple factors contribute to the differences in alcohol binding affinity: the H-bonding with Thr57 and Ser52 (4-5 kcal/mol per H-bond), the desolvation contribution (4-6 kcal/mol for alcohols and 8-10 kcal/mol for water), and the other noncovalent interaction (1.2 kcal/mol per CH(2) group of the alcohol alkyl chain). These results reveal the outstanding potential for using the M05-2X model in calculations of protein-substrate complexes where noncovalent interactions are important.

  16. molecular interactions of the TATA-binding protein

    Indian Academy of Sciences (India)

    Unknown

    Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, ..... Effect of GAL4-VP16 on binding characteristics of yTBP to DNA. Equilibrium dissociation ..... binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar ...

  17. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Directory of Open Access Journals (Sweden)

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  18. Maximizing binding capacity for protein A chromatography.

    Science.gov (United States)

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

    2014-01-01

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

  19. A Crayfish Insulin-like-binding Protein

    Science.gov (United States)

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-01-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  20. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

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

  1. Cation specific binding with protein surface charges.

    Science.gov (United States)

    Hess, Berk; van der Vegt, Nico F A

    2009-08-11

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of "matching water affinities." This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K(+) < Na(+) < Li(+) of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules.

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

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous...

  3. EDTA-insoluble, calcium-binding proteoglycan in bovine bone

    Science.gov (United States)

    Hashimoto, Y.; Lester, G. E.; Caterson, B.; Yamauchi, M.

    1995-01-01

    A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.

  4. EDTA-insoluble, calcium-binding proteoglycan in bovine bone.

    Science.gov (United States)

    Hashimoto, Y; Lester, G E; Caterson, B; Yamauchi, M

    1995-05-01

    A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.

  5. Competitive protein binding assay for piritrexim

    Energy Technology Data Exchange (ETDEWEB)

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. (Wellcome Research Laboratories, Research Triangle Park, NC (USA))

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  6. Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

    NARCIS (Netherlands)

    Snijder, H.J.; Kingma, R.L.; Kalk, K.H.; Dekker, N.; Egmond, M.R.|info:eu-repo/dai/nl/112946593; Dijkstra, B.W.

    2010-01-01

    Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion

  7. Randomized crossover study comparing the phosphate-binding efficacy of calcium ketoglutarate versus calcium carbonate in patients on chronic hemodialysis

    DEFF Research Database (Denmark)

    Bro, S; Rasmussen, R A; Handberg, J

    1998-01-01

    outcome measurements were plasma ionized calcium levels, plasma phosphate levels, plasma intact parathyroid hormone (PTH) levels, requirements for supplemental aluminum-aminoacetate therapy, patient tolerance, and cost of therapy. Nineteen patients on chronic hemodialysis were treated with a dialysate......The objective of the study was to evaluate the phosphate-binding efficacy, side effects, and cost of therapy of calcium ketoglutarate granulate as compared with calcium carbonate tablets in patients on chronic hemodialysis. The study design used was a randomized, crossover open trial, and the main...... lower in the ketoglutarate arm compared with the calcium carbonate arm (4.8+/-0.1 mg/dL v 5.2+/-0.1 mg/dL; P = 0.004), whereas the mean plasma phosphate (4.5+/-0.3 mg/dL v 5.1+/-0.1 mg/dL) and PTH levels (266+/-125 pg/mL v 301+/-148 pg/mL) did not differ significantly between the two treatment arms...

  8. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

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

    1989-01-01

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

  9. An active form of calcium and calmodulin dependant protein kinase ...

    African Journals Online (AJOL)

    The DMI3 gene of the model legume Medicago truncatula encodes a calcium and calmodulin dependent protein kinase (CCaMK) involved in the signalling pathways leading to the establishment of both mycorrhizal and rhizobial root symbiosis. The removal of the auto-inhibitory domain that negatively regulates the kinase ...

  10. Protein mapping of calcium carbonate biominerals by immunogold

    NARCIS (Netherlands)

    Marin, Frédéric; Pokroy, Boaz; Luquet, Gilles; Layrolle, Pierre; de Groot, K.

    2007-01-01

    The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive

  11. Effect of soy protein/animal protein ratio on calcium metabolism of the rat.

    Science.gov (United States)

    Yoon, Gun Ae; Hwang, Hye Jin

    2006-04-01

    This study examined the effects of a ratio of soy protein to animal protein on bone metabolism of rats. Experimental groups were a high soy protein group (200 g of soy protein and 0 g of casein per kilogram of diet; HSoy), a middle soy protein group (100 g of soy protein and 100 g of casein per kilogram of diet; MSoy), a low soy protein group (50 g of soy protein and 150 g of casein per kilogram of diet; LSoy), and a no soy protein group (0 g of soy protein and 200 g of casein per kilogram of diet; NSoy). Calcium excretion and retention, biochemical parametrically related calcium metabolism, and bone mineral density were measured. Statistical analysis was performed with SAS software. Urinary excretion of calcium was significantly high in the LSoy and NSoy groups, and there was no difference in absorption of calcium across experimental groups. Calcium retention was significantly higher in the HSoy and MSoy groups than in the LSoy and NSoy groups. Experimental groups showed no differences in the activity of alkaline phosphatase. The casein group (NSoy) showed a remarkably lower degree of serum osteocalcin concentration. The concentration of deoxypyridinoline in urine showed an increasing tendency, i.e., HSoy and MSoy protein/soy proteins, its concentration increases. Wet weight of the femur appeared to be significantly greater in the MSoy and LSoy groups than in the NSoy group. Ash content of the femur and bone density were highest in the MSoy group, with the ratio of 1:1 between soy protein and animal protein. This study indicated that deoxypyridinoline concentration was lower and the density of osteocalcin was higher in the MSoy group than in the NSoy group, and that calcium retention was high and bone mineral density was the highest in the MSoy group. The ratio of soy to animal protein that seemed to have the most positively significant effect on calcium metabolism was 1:1.

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

    Science.gov (United States)

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

    2010-01-01

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

  13. RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides*

    Science.gov (United States)

    Abdian, Patricia L.; Caramelo, Julio J.; Ausmees, Nora; Zorreguieta, Angeles

    2013-01-01

    In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. PMID:23235153

  14. Comparative footprinting of DNA-binding proteins.

    Science.gov (United States)

    Contreras-Moreira, Bruno; Collado-Vides, Julio

    2006-07-15

    Comparative modelling is a computational method used to tackle a variety of problems in molecular biology and biotechnology. Traditionally it has been applied to model the structure of proteins on their own or bound to small ligands, although more recently it has also been used to model protein-protein interfaces. This work is the first to systematically analyze whether comparative models of protein-DNA complexes could be built and be useful for predicting DNA binding sites. First, we describe the structural and evolutionary conservation of protein-DNA interfaces, and the limits they impose on modelling accuracy. Second, we find that side-chains from contacting residues can be reasonably modeled and therefore used to identify contacting nucleotides. Third, the DNASITE protocol is implemented and different parameters are benchmarked on a set of 85 regulators from Escherichia coli. Results show that comparative footprinting can make useful predictions based solely on structural data, depending primarily on the interface identity with respect to the template used. DNASITE code available on request from the authors.

  15. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  16. Specific association of growth-associated protein 43 with calcium release units in skeletal muscles of lower vertebrates

    Directory of Open Access Journals (Sweden)

    G.A. Caprara

    2014-10-01

    Full Text Available Growth-associated protein 43 (GAP43, is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes, and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species.

  17. EF-hand Ca 2+-binding bioluminescent proteins: effects of mutations and alternative divalent cations

    Science.gov (United States)

    Rowe, Laura; Ensor, Mark; Daunert, Sylvia

    2007-02-01

    Bioluminescent photoproteins, such as aequorin and obelin, are proteins that emit light upon binding calcium. Aequorin and obelin contain four EF-hand domains arranged into a globular structure. The loop region of these EF-hand domains binds calcium by coordinating it in a pentagonal bipyramidal structure with oxygen atoms. The binding of calcium to these EF-hands causes a slight conformational change in the protein, which leads to the oxidation of the internally sequestered chromophore, coelenterazine, producing coelenteramide and CO II. The excited coelenteramide then relaxes radiatively, emitting bioluminescence at 471 nm in aequorin or 491 nm in obelin. Although calcium is the traditional, and generally the most powerful, triggering ligand in this bioluminescence reaction, alternative di- and trivalent cations can also bind to the EF-hand loops and stimulate luminescence. Species capable of this cross-reactivity include: Cd 2+, Ba 2+, Mn 2+, Sr 2+, Mg 2+, and several lanthanides. Magnesium is also known to modulate the bioluminescence of wild-type aequorin, increase its stability, and decrease its aggregation tendency. Both wild-type aequorin and wild-type obelin contain several cysteine residues, aequorin has three and obelin has five. It is believed that these cysteine residues play an important, but as of yet unknown, role in the bioluminescence of these proteins, since mutating most of these residues causes significant loss in bioluminescent activity. In order to explore whether or not these cysteine residues contributed to the specificity of the EF-hand domains for cations we generated four aequorin and obelin mutants and observed their luminescent intensity and decay kinetics by stimulation with calcium, barium, and magnesium. It was found that the cysteine mutations do appear to alter the effects that alternative divalent cations have on the bioluminescence of both aequorin and obelin.

  18. Vitamin D Binding Protein and Bone Health

    Directory of Open Access Journals (Sweden)

    Ishir Bhan

    2014-01-01

    Full Text Available Vitamin D binding protein (DBP is the major carrier protein of 25-hydroxyvitamin D (25(OH D in the circulation, where it may serve roles in maintaining stable levels during times of decreased 25(OH availability and in regulating delivery of 25(OH D to target tissues. Several genetic polymorphisms of DBP have been described that lead to phenotypic changes in the protein that may affect affinity, activity, and concentration. These polymorphisms have been linked with alterations in bone density in several populations. One of the mechanisms by which DBP may alter bone health involves regulating vitamin D bioavailability. DBP-bound vitamin is thought to be relatively unavailable to target tissues, and thus alterations in DBP levels or affinity could lead to changes in vitamin D bioactivity. As a result, functional vitamin D status may differ greatly between individuals with similar total 25(OH D levels. Additionally, DBP may have independent roles on macrophage and osteoclast activation. This review will summarize recent findings about DBP with respect to measures of bone density and health.

  19. A Proteomic Approach to Identification of Plutonium Binding Proteins in Mammalian Cells

    Science.gov (United States)

    Aryal, Baikuntha P.; Paunesku, Tatjana; Woloschak, Gayle E.; He, Chuan; Jensen, Mark P.

    2013-01-01

    Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry were employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu4+-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases. PMID:22146473

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

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

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

  1. Ice-Binding Proteins in Plants

    Directory of Open Access Journals (Sweden)

    Melissa Bredow

    2017-12-01

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

  2. STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

    Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

  3. Calcium-Dependent Protein Kinases in Phytohormone Signaling Pathways

    OpenAIRE

    Wuwu Xu; Wenchao Huang

    2017-01-01

    Calcium-dependent protein kinases (CPKs/CDPKs) are Ca2+-sensors that decode Ca2+ signals into specific physiological responses. Research has reported that CDPKs constitute a large multigene family in various plant species, and play diverse roles in plant growth, development, and stress responses. Although numerous CDPKs have been exhaustively studied, and many of them have been found to be involved in plant hormone biosynthesis and response mechanisms, a comprehensive overview of the manner i...

  4. Protein mapping of calcium carbonate biominerals by immunogold.

    Science.gov (United States)

    Marin, Frédéric; Pokroy, Boaz; Luquet, Gilles; Layrolle, Pierre; De Groot, Klaas

    2007-05-01

    The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive goal. In this paper, we describe a technique for visualizing shell matrix proteins on the surface of calcium carbonate crystals or within the biominerals. The technique is as follows: freshly broken pieces of biominerals or NaOCl then EDTA-etched polished surfaces are incubated with an antibody elicited against one matrix protein, then with a secondary gold-coupled antibody. After silver enhancement, the samples are subsequently observed with scanning electron microscopy by using back-scattered electron mode. In the present case, the technique is applied to a particular example, the calcitic prisms that compose the outer shell layer of the mediterranean fan mussel Pinna nobilis. One major soluble protein, caspartin, which was identified recently, was partly de novo sequenced after enzymatic digestions. A polyclonal antibody raised against caspartin was used for its localization within and on the prisms. The immunogold localization indicated that caspartin surrounds the calcitic prisms, but is also dispersed within the biominerals. This example illustrates the deep impact of the technique on the definition of intracrystalline versus intercrystalline matrix proteins. Furthermore, it is an important tool for assigning a putative function to a matrix protein of interest.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  6. Influence of acid-soluble proteins from bivalve Siliqua radiata ligaments on calcium carbonate crystal growth

    Science.gov (United States)

    Huang, Zeng-Qiong; Zhang, Gang-Sheng

    2016-08-01

    In vitro biomimetic synthesis of calcium carbonate (CaCO3) in the presence of shell proteins is a heavily researched topic in biomineralization. However, little is known regarding the function of bivalve ligament proteins in the growth of CaCO3 crystals. In this study, using fibrous protein K58 from Siliqua radiata ligaments or coverslips as substrates, we report the results of our study of CaCO3 precipitation in the presence or absence of acid-soluble proteins (ASP) from inner ligament layers. ASP can disturb the controlling function of K58 or a coverslip on the crystalline phase, resulting in the formation of aragonite, calcite, and vaterite. In addition, we identified the following four primary components from ASP by mass spectroscopy: alkaline phosphatase (ALP), ABC transporter, keratin type II cytoskeletal 1 (KRT 1), and phosphate ABC transporter, phosphate-binding protein (PstS). Further analysis revealed that the first three proteins and especially ALP, which is important in bone mineralisation, could affect the polymorphism and morphology of CaCO3 crystals by trapping calcium ions in their domains. Our results indicate that ALP may play an important role in the formation of aragonite in S. radiata ligaments. This paper may facilitate our understanding of the biomineralization process.

  7. Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.

    Science.gov (United States)

    Hymel, L; Maurer, A; Berenski, C; Jung, C Y; Fleischer, S

    1984-04-25

    The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.

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

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

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

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

    Science.gov (United States)

    Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-11-26

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

  10. Clarithromycin and tetracycline binding to soil humic acid in the absence and presence of calcium

    Science.gov (United States)

    Christl, Iso; Ruiz, Mercedes; Schmidt, J. R.; Pedersen, Joel A.

    2017-04-01

    Many organic micropollutants including antibiotics contain positively charged moieties and are present as organic cations or zwitterions at environmentally relevant pH conditions. In this study, we investigated the pH-, ionic strength-, and concentration-dependent binding of the two antibiotics clarithromycin and tetracycline to dissolved humic acid in the absence and presence of Ca2+. The investigated compounds strongly differ in their chemical speciation. Clarithromycin can be present as neutral and cationic species, only. But tetracycline can form cations, zwitterions as well as anions and is able to form various calcium complexes. The pH-dependence of binding to soil humic acid was observed to be strongly linked to the protonation behavior for both antibiotics. The presence of Ca2+ decreased clarithromycin binding to soil humic acid, but increased tetracycline binding with increasing Ca2+ concentration. The experimental observations were well described with the NICA-Donnan model considering the complete aqueous speciation of antibiotics and allowing for binding of cationic and zwitterionic species to soil humic acid. Our results indicate that clarithromycin is subject to competition with Ca2+ for binding to soil humic acid and that the electrostatic interaction of positively charged tetracycline-Ca complexes with humic acid enhances tetracycline binding in presence of Ca2+ rather than the formation of ternary complexes, except at very low tetracycline concentrations. We conclude that for the description of ionizable organic micropollutant binding to dissolved natural organic matter, the complete speciation of both sorbate and sorbent has to be considered.

  11. An explicitly solvated full atomistic model of the cardiac thin filament and application on the calcium binding affinity effects from familial hypertrophic cardiomyopathy linked mutations

    Science.gov (United States)

    Williams, Michael; Schwartz, Steven

    2015-03-01

    The previous version of our cardiac thin filament (CTF) model consisted of the troponin complex (cTn), two coiled-coil dimers of tropomyosin (Tm), and 29 actin units. We now present the newest revision of the model to include explicit solvation. The model was developed to continue our study of genetic mutations in the CTF proteins which are linked to familial hypertrophic cardiomyopathies. Binding of calcium to the cTnC subunit causes subtle conformational changes to propagate through the cTnC to the cTnI subunit which then detaches from actin. Conformational changes propagate through to the cTnT subunit, which allows Tm to move into the open position along actin, leading to muscle contraction. Calcium disassociation allows for the reverse to occur, which results in muscle relaxation. The inclusion of explicit TIP3 water solvation allows for the model to get better individual local solvent to protein interactions; which are important when observing the N-lobe calcium binding pocket of the cTnC. We are able to compare in silica and in vitro experimental results to better understand the physiological effects from mutants, such as the R92L/W and F110V/I of the cTnT, on the calcium binding affinity compared to the wild type.

  12. A Ca2+/calmodulin-binding peroxidase from Euphorbia latex: novel aspects of calcium-hydrogen peroxide cross-talk in the regulation of plant defenses.

    Science.gov (United States)

    Mura, Anna; Medda, Rosaria; Longu, Silvia; Floris, Giovanni; Rinaldi, Andrea C; Padiglia, Alessandra

    2005-11-01

    Calmodulin (CaM) is a ubiquitous Ca(2+) sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca(2+), CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca(2+)/CaM-activated proteins are known to tune the interaction between calcium and H(2)O(2) in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918]. Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters showed unequivocally that ELP is a CaM-binding protein activated by the Ca(2+)/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca(2+)/H(2)O(2)-mediated plant defense system, having both positive and negative effects in regulating H(2)O(2) homeostasis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-01

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

  14. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  15. Conformational Changes of Calmodulin on Calcium and Peptide Binding Monitored by Film Bulk Acoustic Resonators

    Science.gov (United States)

    Nirschl, Martin; Ottl, Johannes; Vörös, Janos

    2011-01-01

    Film bulk acoustic resonators (FBAR) are mass sensitive, label-free biosensors that allow monitoring of the interaction between biomolecules. In this paper we use the FBAR to measure the binding of calcium and the CaMKII peptide to calmodulin. Because the mass of the calcium is too small to be detected, the conformational change caused by the binding process is measured by monitoring the resonant frequency and the motional resistance of the FBAR. The resonant frequency is a measure for the amount of mass coupled to the sensor while the motional resistance is influenced by the viscoelastic properties of the adsorbent. The measured frequency shift during the calcium adsorptions was found to be strongly dependent on the surface concentration of the immobilized calmodulin, which indicates that the measured signal is significantly influenced by the amount of water inside the calmodulin layer. By plotting the measured motional resistance against the frequency shift, a mass adsorption can be distinguished from processes involving measurable conformational changes. With this method three serial processes were identified during the peptide binding. The results show that the FBAR is a promising technology for the label-free measurement of conformational changes. PMID:25585566

  16. Consideration on some hormone binding proteins patterns during pregnancy.

    Science.gov (United States)

    Hassan, M A; Miller, N J; Hamdi, I M; el-Adawi, S A; al-Zaid, M; al-Awqati, M A

    1991-02-01

    Serum concentrations of sex hormone binding globulin, transcortin, thyroxine binding globulin, transthyretin together with retinol binding protein, ceruloplasmin, transferrin and albumin were measured sequentially in pregnant women in order to derive more definite suppositions relating to the prime function of hormone binding proteins. Thus, the fact that except for transthyretin all other specific hormone binding proteins exhibited appreciable but significantly variable increases would suggest: a) the apparent existence of more complex mechanisms regulating protein metabolism during pregnancy than hitherto postulated (i.e. the general notion of an integrated estrogen influence); b) a major and distinctive role for each of the hormone binding proteins is plausible since alterations in hormonal requirements by the fetus as pregnancy progresses can not be provided by the almost constant transplacental transfer rate of the "free" hormone moiety.

  17. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  19. Structure and calcium binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp

    Energy Technology Data Exchange (ETDEWEB)

    Hauk, Pricila; Roman-Ramos, Henrique; Ho, Paulo Lee [Instituto Butantan, Sao Paulo, SP (Brazil). Centro de Biotecnologia; Guzzo, Cristiane R.; Farah, Chuck S. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica. Dept. de Bioquimica

    2009-07-01

    Leptospirosis, caused by the spirochaete Leptospira is an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospira. It is highly immunogenic and has been shown to bind to host extracellular matrix components, including collagens, fibronectin and laminin. In this work we crystallized recombinant LipL32 protein and determined its structure to 2.25 A resolution. Initial phases were determined using the multi-wavelength anomalous dispersion technique with data collected from selenomethionine-containing crystals at the MX2 beamline at the LNLS. The LipL32 monomer is made of a jelly-roll fold core from which protrude several peripheral secondary structures. Some structural features suggested that LipL32 could bind Ca{sup 2+} ions and indeed, spectroscopic data (circular (dichroism. intrinsic tryptophan fluorescence and extrinsic 1-amino-2-anaphthol-4-sulfonic acid fluorescence) confirmed the calcium binding properties of LipL32. (author)

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin......In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current...... with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins...

  1. Interleukin-18 and interleukin-18 Binding Protein

    Directory of Open Access Journals (Sweden)

    Charles eDinarello

    2013-10-01

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

  2. IGF Binding Protein-5 Induces Cell Senescence

    Directory of Open Access Journals (Sweden)

    Fumihiro Sanada

    2018-02-01

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

  3. Informing the Human Plasma Protein Binding of ...

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

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

    Science.gov (United States)

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

    2016-10-20

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

  5. Regulation of cell structure and function by actin-binding proteins: villin's perspective.

    Science.gov (United States)

    Khurana, Seema; George, Sudeep P

    2008-06-18

    Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.

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

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Dogan, Jakob; Jemth, Per

    2014-01-01

    Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activatio...... the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins....

  7. Calcium

    Science.gov (United States)

    ... Turn to calcium-fortified (or "calcium-set") tofu, soy milk, tempeh, soy yogurt, and cooked soybeans (edamame). Calcium-fortified foods. Look for calcium-fortified orange juice, soy or rice milk, breads, and cereal. Beans. You can get decent ...

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

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

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

  9. Quantitative analysis of pheromone-binding protein specificity

    OpenAIRE

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-p...

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

    Directory of Open Access Journals (Sweden)

    Evangelia Petsalaki

    2009-03-01

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

  11. CPEB4 is a cell survival protein retained in the nucleus upon ischemia or endoplasmic reticulum calcium depletion.

    Science.gov (United States)

    Kan, Ming-Chung; Oruganty-Das, Aparna; Cooper-Morgan, Amalene; Jin, Guang; Swanger, Sharon A; Bassell, Gary J; Florman, Harvey; van Leyen, Klaus; Richter, Joel D

    2010-12-01

    The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.

  12. CPEB4 Is a Cell Survival Protein Retained in the Nucleus upon Ischemia or Endoplasmic Reticulum Calcium Depletion ▿ †

    Science.gov (United States)

    Kan, Ming-Chung; Oruganty-Das, Aparna; Cooper-Morgan, Amalene; Jin, Guang; Swanger, Sharon A.; Bassell, Gary J.; Florman, Harvey; van Leyen, Klaus; Richter, Joel D.

    2010-01-01

    The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments. PMID:20937770

  13. Mechanism of nuclear calcium signaling by inositol 1,4,5-trisphosphate produced in the nucleus, nuclear located protein kinase C and cyclic AMP-dependent protein kinase.

    Science.gov (United States)

    Klein, Christian; Malviya, Anant N

    2008-01-01

    Nuclear phospholipase C-gamma 1 can be phosphorylated by nuclear membrane located epidermal growth factor receptor sequel to epidermal growth factor-mediated signaling to the nucleus. The function of mouse liver phospholipase C-gamma 1 is attributed to a 120 kDa protein fragment which has been found to be a proteolytic product of the 150 kDa native nuclear enzyme. The tyrosine-phosphorylated 120 kDa protein band interacts with activated EGFR, binds phosphatidyl-3-OH kinase enhancer, and activates nuclear phosphatidylinositol-3-OH-kinase, and is capable of generating diacylglycerol in response to the epidermal growth factor signal to the nucleus in vivo. Thus a mechanism for nuclear production of inositol-1,4,5-trisphophate is unraveled. Nuclear generated inositol-1,4,5-trisphophate interacts with the inner membrane located inositol-1,4,5-trisphophate receptor and sequesters calcium into the nucleoplasm. Nuclear inositol-1,4,5-trisphophate receptor is phosphorylated by native nuclear protein kinase C which enhances the receptor-ligand interaction. Nuclear calcium-ATPase and inositol-1,3,4,5-tetrakisphophate receptor are located on the outer nuclear membrane, thus facilitating calcium transport into the nuclear envelope lumen either by ATP or inositol-1,3,4,5-tetrakisphophate depending upon the external free calcium concentrations. Nuclear calcium ATPase is phosphorylated by cyclic AMP-dependent protein kinase with enhanced calcium pumping activity. A holistic picture emerges here where tyrosine phosphorylation compliments serine phosphorylation of key moieties regulating nuclear calcium signaling. Evidence are forwarded in favor of proteolysis having a profound implications in nuclear calcium homeostasis in particular and signal transduction in general.

  14. Polyester fibers can be rendered calcium phosphate-binding by surface functionalization with bisphosphonate groups.

    Science.gov (United States)

    Polini, Alessandro; Petre, Daniela Geta; Iafisco, Michele; de Lacerda Schickert, Sonia; Tampieri, Anna; van den Beucken, Jeroen; Leeuwenburgh, Sander C G

    2017-08-01

    Fibers are often used as structural elements to improve the mechanical properties of materials such as brittle ceramic matrices by facilitating the dissipation of energy. However, this energy dissipation is mainly controlled by the interface between the two components, and a poorly designed fiber-matrix interface strongly reduces the efficacy of fiber reinforcement. Here, we present a versatile approach to control the affinity of biocompatible fibers to calcium-containing matrices to maximize the efficacy of reinforcement of calcium phosphates-based bioceramics by means of polymeric fibers. To this end, polyester fibers of tunable length were produced by electrospinning and aminolysis, followed by covalent attachment of alendronate, a bisphosphonate molecule with strong calcium-binding affinity, to the surface of the fibers. The proposed method allowed for selective control over the amount of alendronate conjugation, thereby improving the affinity of polyester fibers toward calcium phosphate bioceramics. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2335-2342, 2017. © 2017 Wiley Periodicals, Inc.

  15. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

    Science.gov (United States)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

    1987-01-01

    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  16. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    Science.gov (United States)

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

  17. Roles of Copper-Binding Proteins in Breast Cancer.

    Science.gov (United States)

    Blockhuys, Stéphanie; Wittung-Stafshede, Pernilla

    2017-04-20

    Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.

  18. Post-Translational Modifications and RNA-Binding Proteins.

    Science.gov (United States)

    Lovci, Michael T; Bengtson, Mario H; Massirer, Katlin B

    RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.

  19. Protein (Cyanobacteria): 428310068 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 5 ... calcium-binding protein Microcoleus sp. PCC 7113 MATINGTNFNDNNTFNGIPFIFRPALNGVVDLSFIFPGIIIDLPDTINGLDGNDILNALNTNDTLNGGAGNDT...LFGNAGNDSLDGGAGNDSLDGGTGNDTLLGQGGADTLIGGSGDDLLDSDTLGSVDTIGDLLDGGAGNDTLRGENGNDTLEGGTDDDSLNGGAGNDSLDGGSGDDSLDGGTGNDT...LLGQGGADTLIGGSGDDLLDSDTLGSVDTIGDLLDGGAGNDTLRGENGNDTLEGGT...GNDSLNGGAGNDSLDGGSGDDSLDGGAGNDTLLGQGGADTLIGGSGNDFLNSDTLGAVDTIGDLLDGGDGNDTLQGEAGNDTLLGGIGNDSLTGDDSSIDFGNDSLSGGDGDDTLNGGAGNDT

  20. Protein (Cyanobacteria): 493576985 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2232:23 ... putative calcium-binding protein Gloeocapsa sp. PCC 73106 MSTLSSITGPLYYNHIDGTDGDDSLVGTSLTDGIYGRAGNDTLEGWGANDT...LAGDEGNDSLIGEDGNDSLMGGSENDTLIGGSGNDYLWGHTGNDFLIAGSGNDTLIAEAGNDTLMGEAGNDLLYGGEGDDTLTGGDGNDTLIANTGNDILTGGEGNDT...LIGGTGNNTITGDTGNDTLTGGEGNDTLMGEAGNDTLIGQGGNDSLEGGDGSDLLTGDIGNDTLSGQAGNDT

  1. Protein (Cyanobacteria): 427729770 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available utative calcium-binding protein Nostoc sp. PCC 7524 MPYIPGSNQNDYLQGTAGDDLIETFAGNDTVNVAITIREFVANGQLIRVPIYEPDDDGSGNDT...IWGGIGSDALSGGIGNDSVNGEADNDTLWGGIGNDTVNGGDGNDIINAHQEYVVFRYEGQDYLDSYLVFDEDPGDDFFYGGNGDDFIYAGNGRDYIDGGAG...LDVLYLNTTAYTSNLTVSFTNAANPGTVSTGTQFTGIEYISVTTGNGHDTINLSAFVTGTEVNSGAGNDLITGGTGGDYLYGDAGNDT...INGGDGNDSLNGGSGNDSLDGGAGNDTLIGGAGNDTLNGGSGIDTASYQTATAAVNVNFSTGTATDGQGGTDTLISIERVIGSKFNDTLIGGVGNDSLNGGAGNDTLNGGTGNDT...ASYQNATAAVNVNLSTGITTDGQGGTDTLISIERVIGSKFNDTLIGGSGNETLEGGSGNDTLNGGSGNDILIGNSGNDSLIGGTGNDTLTGGTG

  2. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C......-induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations....... Stable and highly receptor-specific BRET signals were obtained in tsA cells transfected with Rluc- and GFP2-tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least...

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

    Science.gov (United States)

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

    2017-09-01

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

  4. Modulation of calcium oxalate monohydrate crystallization by citrate through selective binding to atomic steps

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, S R; Wierzbicki, A; Salter, E A; Zepeda, S; Orme, C A; Hoyer, J R; Nancollas, G H; Cody, A M; De Yoreo, J J

    2004-10-19

    The majority of human kidney stones are composed primarily of calcium oxalate monohydrate (COM) crystals. Thus, determining the molecular mechanisms by which urinary constituents modulate calcium oxalate crystallization is crucial for understanding and controlling urolithiassis in humans. A comprehensive molecular-scale view of COM shape modification by citrate, a common urinary constituent, obtained through a combination of in situ atomic force microscopy (AFM) and molecular modeling is now presented. We show that citrate strongly influences the growth morphology and kinetics on the (-101) face but has much lower effect on the (010) face. Moreover, binding energy calculations show that the strength of the citrate-COM interaction is much greater at steps than on terraces and is highly step-specific. The maximum binding energy, -166.5 kJ {center_dot} mol{sup -1}, occurs for the [101] step on the (-101) face. In contrast, the value is only -56.9 kJ {center_dot} mol-1 for the [012] step on the (010) face. The binding energies on the (-101) and (010) terraces are also much smaller, -65.4 and -48.9 kJ {center_dot} mol{sup -1} respectively. All other binding energies lie between these extremes. This high selectivity leads to preferential binding of citrate to the acute [101] atomic steps on the (-101) face. The strong citrate-step interactions on this face leads to pinning of all steps, but the anisotropy in interaction strength results in anisotropic reductions in step kinetics. These anisotropic changes in step kinetics are, in turn, responsible for changes in the shape of macroscopic COM crystals. Thus, the molecular scale growth morphology and the bulk crystal habit in the presence of citrate are similar, and the predictions of molecular simulations are fully consistent with the experimental observations.

  5. The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, Anders A.; Sheppard, P O

    1999-01-01

    The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors...... has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated......-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved...

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

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

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

  7. Identification of IgE-binding proteins in soy lecithin.

    Science.gov (United States)

    Gu, X; Beardslee, T; Zeece, M; Sarath, G; Markwell, J

    2001-11-01

    Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins. Copyright 2001 S. Karger AG, Basel

  8. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  9. Library of binding protein scaffolds (LibBP): a computational platform for selection of binding protein scaffolds.

    Science.gov (United States)

    Hong, Seungpyo; Kim, Dongsup

    2016-06-01

    Developments in biotechnology have enabled the in vitro evolution of binding proteins. The emerging limitations of antibodies in binding protein engineering have led to suggestions for other proteins as alternative binding protein scaffolds. Most of these proteins were selected based on human intuition rather than systematic analysis of the available data. To improve this strategy, we developed a computational framework for finding desirable binding protein scaffolds by utilizing protein structure and sequence information. For each protein, its structure and the sequences of evolutionarily-related proteins were analyzed, and spatially contiguous regions composed of highly variable residues were identified. A large number of proteins have these regions, but leucine rich repeats (LRRs), histidine kinase domains and immunoglobulin domains are predominant among them. The candidates suggested as new binding protein scaffolds include histidine kinase, LRR, titin and pentapeptide repeat protein. The database and web-service are accessible via http://bcbl.kaist.ac.kr/LibBP CONTACT: kds@kaist.ac.krSupplementary data: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

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

  11. A linear model for transcription factor binding affinity prediction in protein binding microarrays.

    Directory of Open Access Journals (Sweden)

    Matti Annala

    Full Text Available Protein binding microarrays (PBM are a high throughput technology used to characterize protein-DNA binding. The arrays measure a protein's affinity toward thousands of double-stranded DNA sequences at once, producing a comprehensive binding specificity catalog. We present a linear model for predicting the binding affinity of a protein toward DNA sequences based on PBM data. Our model represents the measured intensity of an individual probe as a sum of the binding affinity contributions of the probe's subsequences. These subsequences characterize a DNA binding motif and can be used to predict the intensity of protein binding against arbitrary DNA sequences. Our method was the best performer in the Dialogue for Reverse Engineering Assessments and Methods 5 (DREAM5 transcription factor/DNA motif recognition challenge. For the DREAM5 bonus challenge, we also developed an approach for the identification of transcription factors based on their PBM binding profiles. Our approach for TF identification achieved the best performance in the bonus challenge.

  12. A method to assay penicillin-binding proteins.

    Science.gov (United States)

    Pucci, Michael J; Dougherty, Thomas J

    2008-01-01

    Key enzymes that assemble the bacterial cell wall are also the target of the Beta-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiology. Methods to label and measure penicillin binding proteins in two prototypical organisms, a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus), are described. The methods discussed include identifying penicillin-binding proteins in both intact cells (in vivo measurements) and isolated cell membranes.

  13. Structural Changes in the Lectin Domain of CD23, the Low-Affinity IgE Receptor, upon Calcium Binding

    Energy Technology Data Exchange (ETDEWEB)

    Wurzburg, Beth A.; Tarchevskaya, Svetlana S.; Jardetzky, Theodore S. (NWU)

    2010-03-08

    CD23, the low-affinity receptor for IgE (Fc{var_epsilon}RII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca{sup 2+}. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca{sup 2+} bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

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

    Directory of Open Access Journals (Sweden)

    Andreas Hoffmann

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

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

    Science.gov (United States)

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

    2011-01-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

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

    Science.gov (United States)

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

    2012-02-01

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

  17. Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus

    Science.gov (United States)

    Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

    2012-01-01

    Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus. PMID:22247597

  18. Roles of phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle twitch dynamics.

    Science.gov (United States)

    Tong, Carl W; Gaffin, Robert D; Zawieja, David C; Muthuchamy, Mariappan

    2004-08-01

    A normal heart increases its contractile force with increasing heart rate. Although calcium handling and myofibrillar proteins have been implicated in maintaining this positive force-frequency relationship (FFR), the exact mechanisms by which it occurs have not been addressed. In this study, we have developed an analytical method to define the calcium-force loop data, which characterizes the function of the contractile proteins in response to calcium that is independent of the calcium handling proteins. Results demonstrate that increasing the stimulation frequency causes increased force production per unit calcium concentration and decreased frequency-dependent calcium sensitivity during the relaxation phase. We hypothesize that phosphorylation of myosin binding protein-C (MyBP-C) and troponin I (TnI) acts coordinately to change the rates of force generation and relaxation, respectively. To test this hypothesis, we performed simultaneous calcium and force measurements on stimulated intact mouse papillary bundles before and after inhibition of MyBP-C and TnI phosphorylation using the calcium/calmodulin kinase II (CaMK2) inhibitor autocamtide-2 related inhibitory peptide, or the protein kinase A (PKA) inhibitor 14-22 amide. CaMK2 inhibition reduced both MyBP-C and TnI phosphorylation and decreased active force without changing the magnitude of the [Ca(2+)](i) transient. This reduced the normalized change in force per change in calcium by 19-39%. Data analyses demonstrated that CaMK2 inhibition changed the myofilament characteristics via a crossbridge feedback mechanism. These results strongly suggest that the phosphorylation of MyBP-C and TnI contributes significantly to the rates of force development and relaxation.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  20. Effects of the binding of calcium ions on the structure and dynamics of the ΦX174 virus investigated using molecular dynamics.

    Science.gov (United States)

    Pina, Jason E; Lee, Kuo Hao; Ytreberg, F Marty

    2012-06-01

    It is known that the presence of calcium ions (Ca(2 + )) is necessary for the enterobacterial virus ΦX174 to inject its DNA into the host cell, and that some mutations in the major capsid proteins lead to better survivability at higher temperatures. Our goal in the current study is to determine the physical changes in both the wild-type and mutant virus due to the binding of Ca(2 + ). Thus, we performed molecular dynamics simulations of the ΦX174 major capsid protein complex with and without Ca(2 + ) bound. Our results show that binding of Ca(2 + ) leads to energetic and dynamical changes in the virus proteins. In particular, the results suggest that binding of Ca(2 + ) is energetically favorable and that the mutation leads to increased fluctuations of the protein complex (especially with the calcium ions bound to the complex), which may increase the rate of genome packaging and ejection for ΦX174.

  1. Involvement of SNP marker located on the Calcium binding protein ...

    African Journals Online (AJOL)

    Olive trees importance is mainly due to the economic and health benefits, especially in the Mediterranean basin. Unfortunately, to enhance productivity and quality of olive oil, the study of both molecular and phenotypic characterizations of olive cultivars is crucial. We consider the analysis of 14 Tunisian olive cultivars of ...

  2. The Art of Compiling Protein Binding Site Ensembles.

    Science.gov (United States)

    Bietz, Stefan; Fährrolfes, Rainer; Rarey, Matthias

    2016-12-01

    Structure-based drug design starts with the collection, preparation, and initial analysis of protein structures. With more than 115,000 structures publically available in the Protein Data Bank (PDB), fully automated processes reliably performing these important preprocessing steps are needed. Several tools are available for these tasks, however, most of them do not address the special needs of scientists interested in protein-ligand interactions. In this paper, we summarize our research activities towards an automated processing pipeline from raw PDB data towards ready-to-use protein binding site ensembles. Starting from a single protein structure, the pipeline covers the following phases: Extracting structurally related binding sites from the PDB, aligning disconnected binding site sequences, resolving tautomeric forms and protonation, orienting hydrogens and flippable side-chains, structurally aligning the multitude of binding sites, and performing a reasonable reduction of ensemble structures. The pipeline, named SIENA, creates protein-structural ensembles for the analysis of protein flexibility, molecular design efforts like docking or de novo design within seconds. For the first time, we are able to process the whole PDB in order to create a large collection of protein binding site ensembles. SIENA is available as part of the ZBH ProteinsPlus webserver under http://proteinsplus.zbh.uni-hamburg.de. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Pleiotropic virulence factor - Streptococcus pyogenes fibronectin-binding proteins.

    Science.gov (United States)

    Yamaguchi, Masaya; Terao, Yutaka; Kawabata, Shigetada

    2013-04-01

    Streptococcus pyogenes causes a broad spectrum of infectious diseases, including pharyngitis, skin infections and invasive necrotizing fasciitis. The initial phase of infection involves colonization, followed by intimate contact with the host cells, thus promoting bacterial uptake by them. S. pyogenes recognizes fibronectin (Fn) through its own Fn-binding proteins to obtain access to epithelial and endothelial cells in host tissue. Fn-binding proteins bind to Fn to form a bridge to α5 β1 -integrins, which leads to rearrangement of cytoskeletal actin in host cells and uptake of invading S. pyogenes. Recently, several structural analyses of the invasion mechanism showed molecular interactions by which Fn converts from a compact plasma protein to a fibrillar component of the extracellular matrix. After colonization, S. pyogenes must evade the host innate immune system to spread into blood vessels and deeper organs. Some Fn-binding proteins contribute to evasion of host innate immunity, such as the complement system and phagocytosis. In addition, Fn-binding proteins have received focus as non-M protein vaccine candidates, because of their localization and conservation among different M serotypes.Here, we review the roles of Fn-binding proteins in the pathogenesis and speculate regarding possible vaccine antigen candidates. © 2012 Blackwell Publishing Ltd.

  4. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case.

    Science.gov (United States)

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-11-21

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features.

  5. The interaction of membrane DNA-binding protein with DNA

    Science.gov (United States)

    Gabrielyan, A. G.; Arakhelyan, H. H.; Zakharyan, R. A.

    1994-07-01

    A 31-kDa protein specifically binding to double-stranded DNA (ds-DNA) was isolated from plasmatic membranes of rat liver cells by means of affinity chromatography and high performance liquid chromatography (HPLC). Some of the properties of this protein were determined. Judging by the UV and circular dichroism spectroscopic data, the protein forms a complex with DNA, stabilizing its native structure, mainly in the regions rich in AT pairs. The 31-kDa protein-pAO3 plasmid DNA binding constant was determined by nitrocellulose filter analysis of protein labelled DNA complexes. The results obtained correspond to cooperative binding with DNA molecules of extended interacting ligands, having AT specificity. A possible role of the 31-kDa protein in DNA transmembrane transition processes is discussed.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  7. RNA-binding specificity of Y-box protein 1.

    Science.gov (United States)

    Dong, Jinjiang; Akcakanat, Argun; Stivers, David N; Zhang, Jiexin; Kim, Doyil; Meric-Bernstam, Funda

    2009-01-01

    Y-box protein 1 (YB-1) is a multifunctional DNA/RNA-binding protein that regulates transcription and translation. The specificity of YB-1's RNA binding and its consequences are unknown. Because expression and subcellular localization of YB-1 have been reported to be important in breast cancer, we determined the specificity and functional impact of YB-1 mRNA-binding in MCF7 breast cancer cells. We used YB-1 antibodies to immunoprecipitate YB-1 and microarray profiling to compare YB-1-bound and total poly(A) RNA. We demonstrated that YB-1 mRNA-binding was preferential. Transcript sequences significantly associated with this binding had high GC content. Selected YB-1 mRNA-binding targets were confirmed by QRT-PCR. However, downregulation of YB-1 levels by siRNA did not affect their RNA or protein expression. Thus, YB-1 has RNA-binding specificity; however, YB-1 binding does not necessarily regulate the stability or translation of its mRNA targets. Further study is needed to determine the functional consequences of selective YB-1 mRNA binding.

  8. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser ...

  9. A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

    Directory of Open Access Journals (Sweden)

    Christine Dorothee Schmeitz

    2013-09-01

    Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

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

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

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

  11. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...

  12. Protein kinase C is a calcium sensor for presynaptic short-term plasticity.

    Science.gov (United States)

    Fioravante, Diasynou; Chu, YunXiang; de Jong, Arthur Ph; Leitges, Michael; Kaeser, Pascal S; Regehr, Wade G

    2014-08-05

    In presynaptic boutons, calcium (Ca(2+)) triggers both neurotransmitter release and short-term synaptic plasticity. Whereas synaptotagmins are known to mediate vesicle fusion through binding of high local Ca(2+) to their C2 domains, the proteins that sense smaller global Ca(2+) increases to produce short-term plasticity have remained elusive. Here, we identify a Ca(2+) sensor for post-tetanic potentiation (PTP), a form of plasticity thought to underlie short-term memory. We find that at the functionally mature calyx of Held synapse the Ca(2+)-dependent protein kinase C isoforms α and β are necessary for PTP, and the expression of PKCβ in PKCαβ double knockout mice rescues PTP. Disruption of Ca(2+) binding to the PKCβ C2 domain specifically prevents PTP without impairing other PKCβ-dependent forms of synaptic enhancement. We conclude that different C2-domain-containing presynaptic proteins are engaged by different Ca(2+) signals, and that Ca(2+) increases evoked by tetanic stimulation are sensed by PKCβ to produce PTP.DOI: http://dx.doi.org/10.7554/eLife.03011.001. Copyright © 2014, Fioravante et al.

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

    African Journals Online (AJOL)

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

  14. How to use not-always-reliable binding site information in protein-protein docking prediction.

    Science.gov (United States)

    Li, Lin; Huang, Yanzhao; Xiao, Yi

    2013-01-01

    In many protein-protein docking algorithms, binding site information is used to help predicting the protein complex structures. Using correct and accurate binding site information can increase protein-protein docking success rate significantly. On the other hand, using wrong binding sites information should lead to a failed prediction, or, at least decrease the success rate. Recently, various successful theoretical methods have been proposed to predict the binding sites of proteins. However, the predicted binding site information is not always reliable, sometimes wrong binding site information could be given. Hence there is a high risk to use the predicted binding site information in current docking algorithms. In this paper, a softly restricting method (SRM) is developed to solve this problem. By utilizing predicted binding site information in a proper way, the SRM algorithm is sensitive to the correct binding site information but insensitive to wrong information, which decreases the risk of using predicted binding site information. This SRM is tested on benchmark 3.0 using purely predicted binding site information. The result shows that when the predicted information is correct, SRM increases the success rate significantly; however, even if the predicted information is completely wrong, SRM only decreases success rate slightly, which indicates that the SRM is suitable for utilizing predicted binding site information.

  15. Anchored clathrate waters bind antifreeze proteins to ice

    OpenAIRE

    Garnham, Christopher P; Campbell, Robert L.; Davies, Peter L.

    2011-01-01

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, t...

  16. Water-binding of protein particles

    NARCIS (Netherlands)

    Peters, J.P.C.M.

    2016-01-01

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

  17. Regulation of CaV2 calcium channels by G protein coupled receptors

    Science.gov (United States)

    Zamponi, Gerald W.; Currie, Kevin P.M.

    2012-01-01

    Voltage gated calcium channels (Ca2+ channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of CaV2 (N- and P/Q-type) Ca2+-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of CaV2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. PMID:23063655

  18. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

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

  19. Engineering a periplasmic binding protein for amino acid sensors with improved binding properties.

    Science.gov (United States)

    Ko, Wooseok; Kim, Sanggil; Lee, Hyun Soo

    2017-10-25

    Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and are involved in the cellular uptake of solutes. In this report, a leucine-binding PBP was engineered to detect l-Leu based on a fluorescence resonance energy transfer (FRET) change upon ligand binding. A fluorescent unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and a yellow fluorescent protein (YFP) was fused with its N-terminus as a FRET acceptor. When CouA was incorporated into position 178, the sensor protein showed a 2.5-fold increase in the FRET ratio. Protein engineering significantly improved its substrate specificity, showing minimal changes in the FRET ratio with the other 19 natural amino acids and d-Leu. Further modification increased the sensitivity of the sensor protein (14-fold) towards l-Leu, and it recognized l-Met as well with moderate binding affinity. Selected mutant sensors were used to measure concentrations of l-Leu in a biological sample (fetal bovine serum) and to determine the optical purity of Leu and Met. This FRET-based sensor design strategy allowed us to easily manipulate the natural receptor to improve its binding affinity and specificity and to recognize other natural molecules, which are not recognized by the wild-type receptor. The design strategy can be applied to other natural receptors, enabling engineering receptors that sense biochemically interesting molecules.

  20. Quantitative analysis of pheromone-binding protein specificity.

    Science.gov (United States)

    Katti, S; Lokhande, N; González, D; Cassill, A; Renthal, R

    2013-02-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins, using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ∼100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ∼200 nM for the silk moth pheromone bombykol and ∼90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed. © 2012 Royal Entomological Society.

  1. Metal-binding proteins as metal pollution indicators

    Energy Technology Data Exchange (ETDEWEB)

    Hennig, H.F.

    1986-03-01

    The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effects on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.

  2. Plasmodium falciparum normocyte binding protein (PfNBP-1) peptides bind specifically to human erythrocytes.

    Science.gov (United States)

    Valbuena, John Jairo; Vera, Ricardo; García, Javier; Puentes, Alvaro; Curtidor, Hernando; Ocampo, Marisol; Urquiza, Mauricio; Rivera, Zuly; Guzmán, Fanny; Torres, Elizabeth; Patarroyo, Manuel Elkin

    2003-07-01

    Plasmodium falciparum normocyte binding protein-1 (PfNBP-1), a Plasmodium vivax RBP-1 orthologue is expressed in the apical merozoite area. PfNBP-1 binds directly to human erythrocyte membrane in a sialic acid-dependent but trypsin-resistant way. Erythrocyte binding assays were done with synthetic peptides covering the sequence reported as PfNBP-1. Two specific erythrocyte high activity binding peptides were found: 101VFINDLDTYQYEYFYEWNQ(120), peptide 26332, and 181NTKETYLKELNKKKMLQNKK(200), peptide 26336. These two peptides' binding was saturable and presenting nanomolar affinity constants. The critical binding residues (those residues underlined and highlighted in bold) were determined by competition assays with glycine-scan analogue peptides. These peptides were able to block merozoite in vitro invasion of erythrocytes.

  3. PYK2: A Calcium-sensitive Protein Tyrosine Kinase Activated in Response to Fertilization of the Zebrafish Oocyte

    Science.gov (United States)

    Sharma, Dipika; Kinsey, William H.

    2012-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  4. Zeatin-binding proteins in barley leaves

    Energy Technology Data Exchange (ETDEWEB)

    Romanov, G.A.; Kulaeva, O.N.; Taryan, V.Y.

    1986-01-01

    Highly labelled tritium-zeatin was used in the work to clarify for the first time a protein factor that is present in cytokinin-sensitive vegetative organs of plants (barley leaves) and which possesses the properties of a cytokinin receptor. Aliquots of tritium-zeatin were mixed with a solution of protein and incubated for several hours in buffer. Following incubation, protein was precipitated by ammonium sulfate at 90% of saturation, and radioactivity of the precipitate was checked in a dioxane scintillator with an efficiency of about 35%. It is shown that the characteristics of interaction of the clarified specific protein sites with cytokinins in regard to a number of criteria correspond to the characteristics expected of receptors of these phytohormones.

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

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

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

  6. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  7. Solid-binding Proteins for Modification of Inorganic Substrates

    Science.gov (United States)

    Coyle, Brandon Laurence

    Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. In this work, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of various proteins to exploit their binding ability. In addition, we conducted a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbon and silicon surfaces. Through these insights, we were able to develop proteins suitable for dispersing graphene flakes and carbon nanotubes in aqueous solutions, while retaining protein activity. Additionally, our investigation into the mechanisms of adhesion for our carbon binding peptides inspired a cheap, disposable protein purification system that is more than 10x cheaper than commonly used His-tag protein purification. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

  8. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key ... [Roy S, Dutta S, Khanna K, Singla S and Sundar D 2012 Prediction of DNA-binding specificity in zinc finger proteins. J. Biosci. .... well as protection from HIV infection (Reynolds, et al. 2003).

  9. Calcium

    Science.gov (United States)

    ... and blood vessels contract and expand, to secrete hormones and enzymes and to send messages through the nervous system. It is important to get plenty of calcium in the foods you eat. Foods rich in calcium include Dairy products such as milk, cheese, and yogurt Leafy, green vegetables Fish with ...

  10. Myosin Binding Protein C Interaction with Actin: CHARACTERIZATION AND MAPPING OF THE BINDING SITE*

    OpenAIRE

    Rybakova, Inna N.; Greaser, Marion L.; Moss, Richard L.

    2010-01-01

    Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and show...

  11. Replacing antibodies: engineering new binding proteins.

    Science.gov (United States)

    Banta, Scott; Dooley, Kevin; Shur, Oren

    2013-01-01

    Nature's reliance on proteins to carry out nearly all biological processes has led to the evolution of biomolecules that exhibit a seemingly endless range of functions. Much research has been devoted toward advancing this process in the laboratory in order to create new proteins with improved or unique capabilities. The protein-engineering field has rapidly evolved from pioneering studies in engineering protein stability and activity to an application-driven powerhouse on the forefront of emerging technologies in biomedical engineering and biotechnology. A classic protein-engineering technique in the medical field has focused on manipulating antibodies and antibody fragments for various applications. New classes of alternative scaffolds have recently challenged this paradigm, and these structures have been successfully engineered for applications including targeted cancer therapy, regulated drug delivery, in vivo imaging, and a host of others. This review aims to capture recent advances in the engineering of nonimmunoglobulin scaffolds as well as some of the applications for these molecular recognition elements in the biomedical field.

  12. Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins.

    Science.gov (United States)

    Murray, M T; Schiller, D L; Franke, W W

    1992-01-01

    Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation.

  13. Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories

    Science.gov (United States)

    Nakayama, S.; Moncrief, N. D.; Kretsinger, R. H.

    1992-01-01

    In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

  14. Building an automated classification of DNA-binding protein domains.

    Science.gov (United States)

    Ponomarenko, Julia V; Bourne, Philip E; Shindyalov, Ilya N

    2002-01-01

    Intensive growth in 3D structure data on DNA-protein complexes as reflected in the Protein Data Bank (PDB) demands new approaches to the annotation and characterization of these data and will lead to a new understanding of critical biological processes involving these data. These data and those from other protein structure classifications will become increasingly important for the modeling of complete proteomes. We propose a fully automated classification of DNA-binding protein domains based on existing 3D-structures from the PDB. The classification, by domain, relies on the Protein Domain Parser (PDP) and the Combinatorial Extension (CE) algorithm for structural alignment. The approach involves the analysis of 3D-interaction patterns in DNA-protein interfaces, assignment of structural domains interacting with DNA, clustering of domains based on structural similarity and DNA-interacting patterns. Comparison with existing resources on describing structural and functional classifications of DNA-binding proteins was used to validate and improve the approach proposed here. In the course of our study we defined a set of criteria and heuristics allowing us to automatically build a biologically meaningful classification and define classes of functionally related protein domains. It was shown that taking into consideration interactions between protein domains and DNA considerably improves the classification accuracy. Our approach provides a high-throughput and up-to-date annotation of DNA-binding protein families which can be found at http://spdc.sdsc.edu.

  15. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

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

    2005-01-01

    described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling......Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including...... endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases...

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-08-28

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

  19. Method for estimating protein binding capacity of polymeric systems

    Directory of Open Access Journals (Sweden)

    Vaibhav Sharma

    2015-01-01

    Full Text Available Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1 synthetic polymer surface characterisation, 2 a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3 an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein on three different polymeric surfaces: silicone, poly(acrylic acid-coated silicone and poly(allylamine-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid and poly(allylamine coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.

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

    Directory of Open Access Journals (Sweden)

    Yoon Sup Choi

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

  1. Characterization of granulations of calcium and apatite in serum as pleomorphic mineralo-protein complexes and as precursors of putative nanobacteria.

    Directory of Open Access Journals (Sweden)

    John D Young

    Full Text Available Calcium and apatite granulations are demonstrated here to form in both human and fetal bovine serum in response to the simple addition of either calcium or phosphate, or a combination of both. These granulations are shown to represent precipitating complexes of protein and hydroxyapatite (HAP that display marked pleomorphism, appearing as round, laminated particles, spindles, and films. These same complexes can be found in normal untreated serum, albeit at much lower amounts, and appear to result from the progressive binding of serum proteins with apatite until reaching saturation, upon which the mineralo-protein complexes precipitate. Chemically and morphologically, these complexes are virtually identical to the so-called nanobacteria (NB implicated in numerous diseases and considered unusual for their small size, pleomorphism, and the presence of HAP. Like NB, serum granulations can seed particles upon transfer to serum-free medium, and their main protein constituents include albumin, complement components 3 and 4A, fetuin-A, and apolipoproteins A1 and B100, as well as other calcium and apatite binding proteins found in the serum. However, these serum mineralo-protein complexes are formed from the direct chemical binding of inorganic and organic phases, bypassing the need for any biological processes, including the long cultivation in cell culture conditions deemed necessary for the demonstration of NB. Thus, these serum granulations may result from physiologically inherent processes that become amplified with calcium phosphate loading or when subjected to culturing in medium. They may be viewed as simple mineralo-protein complexes formed from the deployment of calcification-inhibitory pathways used by the body to cope with excess calcium phosphate so as to prevent unwarranted calcification. Rather than representing novel pathophysiological mechanisms or exotic lifeforms, these results indicate that the entities described earlier as NB most

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

    NARCIS (Netherlands)

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

    1993-01-01

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

  3. Natural history of S-adenosylmethionine-binding proteins

    Directory of Open Access Journals (Sweden)

    Mushegian Arcady R

    2005-10-01

    Full Text Available Abstract Background S-adenosylmethionine is a source of diverse chemical groups used in biosynthesis and modification of virtually every class of biomolecules. The most notable reaction requiring S-adenosylmethionine, transfer of methyl group, is performed by a large class of enzymes, S-adenosylmethionine-dependent methyltransferases, which have been the focus of considerable structure-function studies. Evolutionary trajectories of these enzymes, and especially of other classes of S-adenosylmethionine-binding proteins, nevertheless, remain poorly understood. We addressed this issue by computational comparison of sequences and structures of various S-adenosylmethionine-binding proteins. Results Two widespread folds, Rossmann fold and TIM barrel, have been repeatedly used in evolution for diverse types of S-adenosylmethionine conversion. There were also cases of recruitment of other relatively common folds for S-adenosylmethionine binding. Several classes of proteins have unique unrelated folds, specialized for just one type of chemistry and unified by the theme of internal domain duplications. In several cases, functional divergence is evident, when evolutionarily related enzymes have changed the mode of binding and the type of chemical transformation of S-adenosylmethionine. There are also instances of functional convergence, when biochemically similar processes are performed by drastically different classes of S-adenosylmethionine-binding proteins. Comparison of remote sequence similarities and analysis of phyletic patterns suggests that the last universal common ancestor of cellular life had between 10 and 20 S-adenosylmethionine-binding proteins from at least 5 fold classes, providing for S-adenosylmethionine formation, polyamine biosynthesis, and methylation of several substrates, including nucleic acids and peptide chain release factor. Conclusion We have observed several novel relationships between families that were not known to be

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

    Science.gov (United States)

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

    2017-01-01

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

  5. RNA-Binding Proteins in Female Reproductive Pathologies.

    Science.gov (United States)

    Khalaj, Kasra; Miller, Jessica E; Fenn, Christian R; Ahn, SooHyun; Luna, Rayana L; Symons, Lindsey; Monsanto, Stephany P; Koti, Madhuri; Tayade, Chandrakant

    2017-06-01

    RNA-binding proteins are key regulatory molecules involved primarily in post-transcriptional gene regulation of RNAs. Post-transcriptional gene regulation is critical for adequate cellular growth and survival. Recent reports have shown key interactions between these RNA-binding proteins and other regulatory elements, such as miRNAs and long noncoding RNAs, either enhancing or diminishing their response to RNA stabilization. Many RNA-binding proteins have been reported to play a functional role in mediation of cytokines involved in inflammation and immune dysfunction, and some have been classified as global post-transcriptional regulators of inflammation. The ubiquitous expression of RNA-binding proteins in a wide variety of cell types and their unique mechanisms of degradative action provide evidence that they are involved in reproductive tract pathologies. Aberrant inflammation and immune dysfunction are major contributors to the pathogenesis and disease pathophysiology of many reproductive pathologies, including ovarian and endometrial cancers in the female reproductive tract. Herein, we discuss various RNA-binding proteins and their unique contributions to female reproductive pathologies with a focus on those mediated by aberrant inflammation and immune dysfunction. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

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

  7. Retinoblastoma-binding Protein 1 Has an Interdigitated Double Tudor Domain with DNA Binding Activity*

    Science.gov (United States)

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

    2014-01-01

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10–100 μm; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1. PMID:24379399

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

    Science.gov (United States)

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

    2014-02-21

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

  9. Binding dynamics of single-stranded DNA binding proteins to fluctuating bubbles in breathing DNA

    Energy Technology Data Exchange (ETDEWEB)

    Ambjoernsson, Tobias; Metzler, Ralf [NORDITA-Nordic Institute for Theoretical Physics, Blegdamsvej 17, DK-2100 Copenhagen Oe (Denmark)

    2005-05-25

    We investigate the dynamics of a single local denaturation zone in a DNA molecule, a so-called DNA bubble, in the presence of single-stranded DNA binding proteins (SSBs). In particular, we develop a dynamical description of the process in terms of a two-dimensional master equation for the time evolution of the probability distribution of having a bubble of size m with n bound SSBs, for the case when m and n are the slowest variables in the system. We derive explicit expressions for the equilibrium statistical weights for a given m and n, which depend on the statistical weight u associated with breaking a base-pair interaction, the loop closure exponent c, the cooperativity parameter {sigma}{sub 0}, the SSB size {lambda}, and binding strength {kappa}. These statistical weights determine, through the detailed balance condition, the transfer coefficient in the master equation. For the case of slow and fast binding dynamics the problem can be reduced to one-dimensional master equations. In the latter case, we perform explicitly the adiabatic elimination of the fast variable n. Furthermore, we find that for the case that the loop closure is neglected and the binding dynamics is vanishing (but with arbitrary {sigma}{sub 0}) the eigenvalues and the eigenvectors of the master equation can be obtained analytically, using an orthogonal polynomial approach. We solve the general case numerically (i.e., including SSB binding and the loop closure) as a function of statistical weight u, binding protein size {lambda}, and binding strength {kappa}, and compare to the fast and slow binding limits. In particular, we find that the presence of SSBs in general increases the relaxation time, compared to the case when no binding proteins are present. By tuning the parameters, we can drive the system from regular bubble fluctuation in the absence of SSBs to full denaturation, reflecting experimental and in vivo situations.

  10. Computational comparison of a calcium-dependent jellyfish protein (apoaequorin) and calmodulin-cholesterol in short-term memory maintenance.

    Science.gov (United States)

    Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K

    2017-03-06

    Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca 2+ -binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca 2+ ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca 2+ ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca 2+ -binding sites. Only calmodulin pore-lining regions contain Ca 2+ binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca 2+ channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to

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

    Science.gov (United States)

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

    2017-04-04

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

  12. STRUCTURAL INSIGHT INTO THE FORMIN BINDING PROPERTY OF HUMAN FORMIN BINDING PROTEIN 4

    Directory of Open Access Journals (Sweden)

    Amit Das

    2013-12-01

    Full Text Available Formin mediated dynamic regulation of actin cytoskeleton is involved in numerous cellular processes, organism growth and development and different diseases. The cellular formin-actin network is further fine tuned by different formin binding proteins (FNBPs. Four such FNBPs which were initially discovered as formin poly proline rich FH1 domain binding; were found to contain tandem WW domains as their formin interacting module. Despite the fact that WW domains are the smallest known protein module that is involved in multi-protein interactions, the interaction of formins and WW domain containing FNBPs were rarely explored and their interactions under physiological contexts became doubtful. However different studies continuously provided indications of greater cellular impacts of these interactions. We have used computational techniques (homology modeling and molecular docking to investigate the ligand binding, especially formin binding properties of human FNBP4 WW domains. We have found that the triple β sheet structure of the 1st WW domain of FNBP4 can bind to the proline rich FH1 region of formin mDia1. However the FNBP4 second WW domain was found to be unable to bind to mDia1 FH1 region. Detailed investigation showed the importance of sequence variations of FNBP4 WW domains in specifying their interactions with formins

  13. Calcium

    Science.gov (United States)

    ... from dietary supplements are linked to a greater risk of kidney stones, especially among older adults. But calcium from foods does not appear to cause kidney stones. For most people, other factors (such as not drinking enough fluids) probably have ...

  14. Interactome-Wide Prediction of Protein-Protein Binding Sites Reveals Effects of Protein Sequence Variation in Arabidopsis thaliana

    NARCIS (Netherlands)

    Valentim, F.L.; Neven, F.; Boyen, P.; Dijk, van A.D.J.

    2012-01-01

    The specificity of protein-protein interactions is encoded in those parts of the sequence that compose the binding interface. Therefore, understanding how changes in protein sequence influence interaction specificity, and possibly the phenotype, requires knowing the location of binding sites in

  15. Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen (plasmin fragments Y)

    NARCIS (Netherlands)

    Nieuwenhuizen, W.; Voskuilen, M.; Hermans, J.

    1982-01-01

    The present study was undertaken as a step to delineate further the localization of the calcium-binding sites in fibrinogen and to assess the anticlotting properties of fibrinogen degradation products. To this purpose, fragments Y were prepared by plasmin digestion of human fibrinogen in the

  16. Solvation structure of ice-binding antifreeze proteins

    Science.gov (United States)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

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

  17. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  18. Modularized study of human calcium signalling pathway

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    When there is an extracellular change, cells get the message either by introduction of calcium ions into ... as it precipitates phosphate, the established energy currency of cells. Prolonged high intracellular calcium ... trigger proteins upon binding with free calcium ion(s) change their confirmation to modulate enzymes and ion ...

  19. TAK1-binding protein 1 is a pseudophosphatase

    Science.gov (United States)

    Conner, Sarah H.; Kular, Gursant; Peggie, Mark; Shepherd, Sharon; Schüttelkopf, Alexander W.; Cohen, Philip; Van Aalten, Daan M. F.

    2006-01-01

    TAB1 [TAK1 (transforming growth factor-β-activated kinase 1)-binding protein 1] is one of the regulatory subunits of TAK1, a protein kinase that lies at the head of three pro-inflammatory kinase cascades. In the current study we report the crystal structure of the N-terminal domain of TAB1. Surprisingly, TAB1 possesses a fold closely related to that of the PPM (Mg2+- or Mn2+-dependent protein phosphatase) family as demonstrated by the close structural similarity with protein phosphatase 2Cα. However, we were unable to detect any phosphatase activity for TAB1 using a phosphopeptide or p-nitrophenyl phosphate as substrate. Although the overall protein phosphatase 2Cα fold is conserved in TAB1, detailed structural analyses and mutagenesis studies show that several key residues required for dual metal-binding and catalysis are not present in TAB1, although binding of a single metal is supported by soaking experiments with manganese and isothermal titration calorimetry. Thus, it appears that TAB1 is a ‘pseudophosphatase’, possibly binding to and regulating accessibility of phosphorylated residues on substrates downstream of TAK1 or on the TAK1 complex itself. PMID:16879102

  20. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

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

  1. RBPPred: predicting RNA-binding proteins from sequence using SVM.

    Science.gov (United States)

    Zhang, Xiaoli; Liu, Shiyong

    2017-03-15

    Detection of RNA-binding proteins (RBPs) is essential since the RNA-binding proteins play critical roles in post-transcriptional regulation and have diverse roles in various biological processes. Moreover, identifying RBPs by computational prediction is much more efficient than experimental methods and may have guiding significance on the experiment design. In this study, we present the RBPPred (an RNA-binding protein predictor), a new method based on the support vector machine, to predict whether a protein binds RNAs, based on a comprehensive feature representation. By integrating the physicochemical properties with the evolutionary information of protein sequences, the new approach RBPPred performed much better than state-of-the-art methods. The results show that RBPPred correctly predicted 83% of 2780 RBPs and 96% out of 7093 non-RBPs with MCC of 0.808 using the 10-fold cross validation. Furthermore, we achieved a sensitivity of 84%, specificity of 97% and MCC of 0.788 on the testing set of human proteome. In addition we tested the capability of RBPPred to identify new RBPs, which further confirmed the practicability and predictability of the method. RBPPred program can be accessed at: http://rnabinding.com/RBPPred.html . liushiyong@gmail.com. Supplementary data are available at Bioinformatics online.

  2. Lamins and lamin-binding proteins in functional chromatin organization.

    Science.gov (United States)

    Gotzmann, J; Foisner, R

    1999-01-01

    Lamins are the major components of the nuclear lamina, a two-dimensional filamentous network at the periphery of the nucleus in higher eukaryotes, directly underlying the inner nuclear membrane. Several integral proteins of the inner nuclear membrane bind to lamins and may link the nuclear membrane to the core lamina network. The lamins and the lamin-binding proteins lamina-associated polypeptide (LAP)2beta and lamin B receptor (LBR) have been described to bind to DNA or to interact with chromatin via histones, BAF-1, and HP1 chromodomain proteins, respectively, and may provide anchorage sites for chromatin fibers at the nuclear periphery. In addition, lamin A structures on intranuclear filaments, or lamin B in replication foci have been described in the nuclear interior, but their specific roles remain unclear. An isoform of the LAP2 protein family, LAP2alpha, has been found to colocalize with A-type lamins in the nucleoplasm and might be involved in intranuclear structure organization. In the course of cell-cycle-dependent dynamics of the nucleus in higher eukaryotes, lamins as well as lamin-binding proteins seem to possess important functions during various steps of post-mitotic nuclear reassembly, including cross-linking of chromatides, nuclear membrane targeting, nuclear lamina assembly, and the formation of a replication-competent nucleus.

  3. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

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

    2017-01-01

    The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of amino...... of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-determining residues. These findings lay the foundation...

  4. Intake of protein, calcium and sodium in public child day care centers

    Directory of Open Access Journals (Sweden)

    Giovana Longo-Silva

    2014-06-01

    Full Text Available OBJECTIVE:To assess calcium, protein and sodium intake, of children that attend public day-care centers and to compare it with the recommended one.METHODS:Cross-sectional descriptive study in seven public day care centers of São Paulo city, Southeast Brazil, which enrolled 366 children between 12 and 36 months of age. The data collection occurred between September and December 2010. Each day care center was evaluated for three non-consecutive days, totaling 42 days and 210 meals. Dietary intake was assessed by a direct food weighing method. For the nutritional calculation, DietWin(r Profissional 2.0 was used, and the adequacy was calculated according to the recommendations of the National School Feeding Program for energy, protein, calcium and sodium. The calcium/protein relation was also calculated, as well as calcium density (mg/1,000kcal.RESULTS: The energy (406.4kcal, protein (18.2g and calcium (207.6mg consumption did not reach the recommended values ​​in all the evaluated day care centers. Sodium intake exceeded up to three times the recommendation. The calcium/protein ratio of 11.7mg/g was less than the adequate one (20mg/g.CONCLUSIONS: There was inadequacy of calcium, protein and sodium dietary intake, in children attending public day-care centers.

  5. Comprehensive behavioral analysis of calcium/calmodulin-dependent protein kinase IV knockout mice.

    Directory of Open Access Journals (Sweden)

    Keizo Takao

    Full Text Available Calcium-calmodulin dependent protein kinase IV (CaMKIV is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency.

  6. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    Science.gov (United States)

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-06-04

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

  8. Structure of Osh3 reveals a conserved mode of phosphoinositide binding in oxysterol-binding proteins.

    Science.gov (United States)

    Tong, Junsen; Yang, Huiseon; Yang, Hongyuan; Eom, Soo Hyun; Im, Young Jun

    2013-07-02

    The oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans, and implicated in the regulation of lipid homeostasis and in signaling pathways. Saccharomyces cerevisiae has seven ORPs (Osh1-Osh7) that share one unknown essential function. Here, we report the 1.5-2.3 Å structures of the PH domain and ORD (OSBP-related domain) of yeast Osh3 in apo-form or in complex with phosphatidylinositol 4-phosphate (PI[4]P). Osh3 recognizes PI(4)P by the highly conserved residues in the tunnel of ORD whereas it lacks sterol binding due to the narrow hydrophobic tunnel. Yeast complementation tests suggest that PI(4)P binding to PH and ORD is essential for function. This study suggests that the unifying feature in all ORP homologs is the binding of PI(4)P to ORD and sterol binding is additional to certain homologs. Structural modeling of full-length Osh3 is consistent with the concept that Osh3 is a lipid transfer protein or regulator in membrane contact sites. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Plasmonic nanosensors for simultaneous quantification of multiple protein-protein binding affinities.

    Science.gov (United States)

    Ahijado-Guzmán, Rubén; Prasad, Janak; Rosman, Christina; Henkel, Andreas; Tome, Lydia; Schneider, Dirk; Rivas, Germán; Sönnichsen, Carsten

    2014-10-08

    Most of current techniques used for the quantification of protein-protein interactions require the analysis of one pair of binding partners at a time. Herein we present a label-free, simple, fast, and cost-effective route to characterize binding affinities between multiple macromolecular partners simultaneously, using optical dark-field spectroscopy and individual protein-functionalized gold nanorods as sensing elements. Our NanoSPR method could easily become a simple and standard tool in biological, biochemical, and medical laboratories.

  10. Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1.

    Science.gov (United States)

    Child, Matthew A; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A; Boothroyd, John C; Reese, Michael L; Bogyo, Matthew

    2017-02-28

    Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-15

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

  12. Cooperative interactions between odorant-binding proteins of Anopheles gambiae.

    Science.gov (United States)

    Qiao, Huili; He, Xiaoli; Schymura, Danuta; Ban, Liping; Field, Linda; Dani, Francesca Romana; Michelucci, Elena; Caputo, Beniamino; della Torre, Alessandra; Iatrou, Kostas; Zhou, Jing-Jiang; Krieger, Jürgen; Pelosi, Paolo

    2011-05-01

    To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.

  13. RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins

    Science.gov (United States)

    Lambert, Nicole; Robertson, Alex; Jangi, Mohini; McGeary, Sean; Sharp, Phillip A.; Burge, Christopher B.

    2014-01-01

    Summary Specific protein-RNA interactions guide post-transcriptional gene regulation. Here we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1 and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity. PMID:24837674

  14. Flexibility of PCNA-protein interface accommodates differential binding partners.

    Science.gov (United States)

    Pedley, Anthony M; Lill, Markus A; Davisson, V Jo

    2014-01-01

    The expanding roles of PCNA in functional assembly of DNA replication and repair complexes motivated investigation of the structural and dynamic properties guiding specificity of PCNA-protein interactions. A series of biochemical and computational analyses were combined to evaluate the PIP Box recognition features impacting complex formation. The results indicate subtle differences in topological and molecular descriptors distinguishing both affinity and stoichiometry of binding among PCNA-peptide complexes through cooperative effects. These features were validated using peptide mimics of p85α and Akt, two previously unreported PCNA binding partners. This study characterizes for the first time a reverse PIP Box interaction with PCNA. Small molecule ligand binding at the PIP Box interaction site confirmed the adaptive nature of the protein in dictating overall shape and implicates allosterism in transmitting biological effects.

  15. Flexibility of PCNA-protein interface accommodates differential binding partners.

    Directory of Open Access Journals (Sweden)

    Anthony M Pedley

    Full Text Available The expanding roles of PCNA in functional assembly of DNA replication and repair complexes motivated investigation of the structural and dynamic properties guiding specificity of PCNA-protein interactions. A series of biochemical and computational analyses were combined to evaluate the PIP Box recognition features impacting complex formation. The results indicate subtle differences in topological and molecular descriptors distinguishing both affinity and stoichiometry of binding among PCNA-peptide complexes through cooperative effects. These features were validated using peptide mimics of p85α and Akt, two previously unreported PCNA binding partners. This study characterizes for the first time a reverse PIP Box interaction with PCNA. Small molecule ligand binding at the PIP Box interaction site confirmed the adaptive nature of the protein in dictating overall shape and implicates allosterism in transmitting biological effects.

  16. T3: Targeted Proteomics of DNA-Binding Proteins

    Science.gov (United States)

    Nagore, Linda I.; Jarrett, Harry W.

    2015-01-01

    A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TF) while providing a route for high throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TF by combining nuclear extract with a specific DNA sequence and immobilizing the DNA-protein complex on a PVP-coated MALDI plate. Any unbound proteins are washed away and further processed for analysis in a MALDI-TOF/TOF mass spectrometer. Enrichment on a PVP-coated plate gives the unique advantage of purification, enzymatic digestion and analysis on a single platform. The method is termed T3 as it combines Targeted purification on a Target plate with Targeted proteomics. Validation was achieved in model experiments with a chimeric fusion protein, green fluorescent protein-CAAT enhancer binding protein (GFP-C/EBP) with an oligonucleotide containing the CAAT sequence. Both domains were identified with an expectation value of less than 10−15 and over 15% sequence coverage. The same oligonucleotide mixed with HEK293 cell nuclear extract allowed the unambiguous identification of native human C/EBP alpha with 24.3% sequence coverage. PMID:25644705

  17. Isolation and characterization of calcium binding glycoproteins of cardiac sarcolemmal vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Michalak, M.; Fliegel, L.; Wlasichuk, K. (Univ. of Alberta, Edmonton (Canada))

    1990-04-05

    Two major Ca2(+)-binding glycoproteins Mr 120,000 and 100,000 were isolated from 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid -solubilized bovine heart sarcolemma membrane. Peroxidase-conjugated concanavalin A and wheat germ agglutinin lectins bind strongly to the isolated 120- and 100-kDa glycoproteins. Treatment with endoglycosidase F resulted in conversion of the 120-kDa glycoprotein to a form migrating at about 97 kDa. Treatment of the 100-kDa band with endoglycosidase F produced form of about 80 kDa. Endoglycosidase H digestion removes only 5% of the mass of both glycoproteins. the carbohydrate structure of both glycoproteins, is therefore, predicted to be at least 75% complex structure and 25% high mannose or hybrid structure. The 120- and 100-kDa glycoproteins are the major Ca2(+)-binding proteins in the sarcolemma membranes. Intact and endoglycosidase-treated glycoproteins bind 45Ca2+ as analyzed by a 45Ca2+ overlay technique. Using polyclonal antibodies, the 120- and 100-kDa glycoproteins were identified in muscle plasma membranes (ventricles, atria, and uterus smooth muscle). They were, however, not present in non-muscle tissues such as pancreas, liver, and kidney. The 120- and 100-kDa glycoproteins appear to be homologous molecules as judged by their similar V8 protease peptide maps, cross-reactivity with polyclonal antibody, and other physicochemical properties.

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

    Indian Academy of Sciences (India)

    Madhu

    The transcription factor cyclic AMP response element binding protein (CREB) and the neurotrophin brain-derived neurotrophic factor (BDNF) are targets of diverse classes of antidepressants and are known to be regulated in animal models and in patients suffering from depression. Given their role in neuronal plasticity, ...

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

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

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

  20. Controlling transcription fidelity via TATA-binding protein dynamics

    NARCIS (Netherlands)

    Koster, M.J.E.

    2015-01-01

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

  1. QSAR models for the prediction of plasma protein binding.

    Science.gov (United States)

    Ghafourian, Taravat; Amin, Zeshan

    2013-01-01

    The prediction of plasma protein binding (ppb) is of paramount importance in the pharmacokinetics characterization of drugs, as it causes significant changes in volume of distribution, clearance and drug half life. This study utilized Quantitative Structure - Activity Relationships (QSAR) for the prediction of plasma protein binding. Protein binding values for 794 compounds were collated from literature. The data was partitioned into a training set of 662 compounds and an external validation set of 132 compounds. Physicochemical and molecular descriptors were calculated for each compound using ACD labs/logD, MOE (Chemical Computing Group) and Symyx QSAR software packages. Several data mining tools were employed for the construction of models. These included stepwise regression analysis, Classification and Regression Trees (CART), Boosted trees and Random Forest. Several predictive models were identified; however, one model in particular produced significantly superior prediction accuracy for the external validation set as measured using mean absolute error and correlation coefficient. The selected model was a boosted regression tree model which had the mean absolute error for training set of 13.25 and for validation set of 14.96. Plasma protein binding can be modeled using simple regression trees or multiple linear regressions with reasonable model accuracies. These interpretable models were able to identify the governing molecular factors for a high ppb that included hydrophobicity, van der Waals surface area parameters, and aromaticity. On the other hand, the more complicated ensemble method of boosted regression trees produced the most accurate ppb estimations for the external validation set.

  2. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    Science.gov (United States)

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

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

    African Journals Online (AJOL)

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

  4. Pathogenesis of ceftriaxone-associated biliary sludge. In vitro studies of calcium-ceftriaxone binding and solubility.

    Science.gov (United States)

    Shiffman, M L; Keith, F B; Moore, E W

    1990-12-01

    Ceftriaxone, a semisynthetic third-generation cephalosporin, has recently been associated with biliary sludge formation. Analysis of the biliary concretions induced by this agent shows a calcium salt of ceftriaxone. The present in vitro studies were undertaken to provide insight into the pathogenesis of ceftriaxone-associated biliary sludge formation by evaluating possible interactions that may exist between calcium, bile salts, and ceftriaxone. Ceftriaxone possessed high calcium-binding affinity. The formation constant for the calcium ceftriaxone salt at 37 degrees C was about 157.3 L/mol; stoichiometry of the salt was 1:1, i.e., calcium ceftriaxone. The calcium-binding property of ceftriaxone was observed to be additive to that of taurocholate in mixed taurocholate-ceftriaxone solutions. Although the solubility product constant for calcium ceftriaxone was only 1.62 x 10(-6) mol/L2, marked metastability was observed; neither visible nor microscopic precipitates developed until the [Ca2+] x [ceftriaxone] ion product exceeded the solubility product constant by a factor of 10.4. Metastability of the calcium ceftriaxone salt was also observed in human gallbladder bile in vitro. Estimates of human biliary calcium ceftriaxone solubility in vivo were than calculated from previously-reported values for biliary [Ca2+], [ceftriaxone], and from the solubility product constant as defined in this study. Calculated saturation indices for calcium-ceftriaxone in human bile generally increased (corresponding to a decrease in solubility) with increasing ceftriaxone dose. At doses less than or equal to 1 g, saturation index was well within the metastable range of this calcium-salt. However, at doses greater than or equal to 2 g, the saturation index surpassed the metastable limit. Under these conditions, precipitation of ceftriaxone could occur. It was concluded that the development of ceftriaxone-induced biliary sludge is a solubility problem that occurs in patients receiving high

  5. Damage-specific DNA-binding proteins from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1

  6. High calcium and dobutamine positive inotropy in the perfused mouse heart: myofilament calcium responsiveness, energetic economy, and effects of protein kinase C inhibition

    Directory of Open Access Journals (Sweden)

    Du Congwu

    2001-08-01

    Full Text Available Abstract Background In perfused hearts, high calcium-induced inotropy results in less developed pressure relative to myocardial oxygen consumption compared to the β-adrenergic agonist dobutamine. Calcium handling is an important determinant of myocardial oxygen consumption. Therefore, we hypothesized that this phenomenon was due to reduced myofilament responsiveness to calcium, related to protein kinase C activation. Results Developed pressure was significantly higher with dobutamine compared to high perfusate calcium of 3.5 mM (73 ± 10 vs 63 ± 10 mmHg, p Conclusions By measuring intracellular calcium, developed pressures and myocardial oxygen consumption in perfused mouse hearts, these results demonstrate that high perfusate calcium positive inotropy compared to dobutamine results in reduced myofilament responsiveness to intracellular calcium, which is associated with energetic inefficiency and evidence of protein kinase C activation.

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

    Science.gov (United States)

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

    2017-12-01

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

  8. Controlled substitution of soy protein for meat protein: effects on calcium retention, bone, and cardiovascular health indices in postmenopausal women.

    Science.gov (United States)

    Roughead, Zamzam K; Hunt, Janet R; Johnson, Luann K; Badger, Thomas M; Lykken, Glenn I

    2005-01-01

    In a controlled feeding study, the effects of substituting 25 g soy protein for meat on calcium retention and bone biomarkers were determined. Postmenopausal women (n = 13) ate two diets that were similar, except that, in one diet, 25 g high-isoflavone soy protein (SOY) was substituted for an equivalent amount of meat protein (control diet), for 7 wk each in a randomized crossover design. After 3 wk of equilibration, calcium retention was measured by labeling the 2-d menu with (47)Ca, followed by whole-body counting for 28 d. Urinary calcium and renal acid excretion were measured at wk 3, 5, and 7. Biomarkers of bone and cardiovascular health were measured at the beginning and end of each diet. Calcium was similarly retained during the control and SOY diets (d 28, percent dose, mean +/- pooled sd: 14.1 and 14.0 +/- 1.6, respectively). Despite a 15-20% lower renal acid excretion during the SOY diet, urinary calcium loss was unaffected by diet. Diet also did not affect any of the indicators of bone or cardiovascular health. Substitution of 25 g high isoflavone soy protein for meat, in the presence of typical calcium intakes, did not improve or impair calcium retention or indicators of bone and cardiovascular health in postmenopausal women.

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    the highly conserved 60 amino acid homeodomain. This peptide antiserum recognized a protein species of molecular weight 63,000 in immunoblots of nuclear extracts obtained from several tumor cell lines. The predominant molecular weight 63,000 nuclear protein recognized by the peptide antiserum...... the same patients exhibited little immunoreactivity. Both the peptide antiserum and the polyclonal antiserum against the native protein immunoblotted a molecular weight 63,000 protein in nuclear extracts of tumor tissue, but not significantly in extracts of normal tissue. At the molecular level......Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...

  10. Lactoferrin, an iron-binding protein in neutrophilic leukocytes.

    Science.gov (United States)

    Masson, P L; Heremans, J F; Schonne, E

    1969-09-01

    Lactoferrin, an iron-binding protein previously shown to occur in many external secretions, is identified as one of the major proteins present in human and guinea pig neutrophilic polymorphonuclear leukocytes. The identification of this protein in leukocyte extracts was based upon a comparison of its electrophoretic, antigenic, and iron-combining properties with the corresponding properties of the same protein isolated from human and guinea pig milk. Immunochemical quantitations showed that lactoferrin occurs in human neutrophilic leukocytes at the concentration of 3 microg per 10(6) cells. Tissue cultures from guinea pig bone marrow and spleen actively synthesized the protein, as shown both by net production of lactoferrin and incorporation of labeled amino acids into the protein. Immunohistochemical data indicate that lactoferrin first appears in myeloid cells at the stage of the promyelocyte.

  11. Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes. Influence of anti-collagen-antibodies

    DEFF Research Database (Denmark)

    Holmskov, U; Haas, Henning de; Teisner, B

    1992-01-01

    G eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes......G with serum at 37 degrees C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (greater than 600 kDa). Binding of Ig......G to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37 degrees C. The reactive Ig...

  12. Lead(II) Binding in Natural and Artificial Proteins.

    Science.gov (United States)

    Cangelosi, Virginia; Ruckthong, Leela; Pecoraro, Vincent L

    2017-04-10

    This article describes recent attempts to understand the biological chemistry of lead using a synthetic biology approach. Lead binds to a variety of different biomolecules ranging from enzymes to regulatory and signaling proteins to bone matrix. We have focused on the interactions of this element in thiolate-rich sites that are found in metalloregulatory proteins such as Pbr, Znt, and CadC and in enzymes such as δ-aminolevulinic acid dehydratase (ALAD). In these proteins, Pb(II) is often found as a homoleptic and hemidirectic Pb(II)(SR)3- complex. Using first principles of biophysics, we have developed relatively short peptides that can associate into three-stranded coiled coils (3SCCs), in which a cysteine group is incorporated into the hydrophobic core to generate a (cysteine)3 binding site. We describe how lead may be sequestered into these sites, the characteristic spectral features may be observed for such systems and we provide crystallographic insight on metal binding. The Pb(II)(SR)3- that is revealed within these α-helical assemblies forms a trigonal pyramidal structure (having an endo orientation) with distinct conformations than are also found in natural proteins (having an exo conformation). This structural insight, combined with 207Pb NMR spectroscopy, suggests that while Pb(II) prefers hemidirected Pb(II)(SR)3- scaffolds regardless of the protein fold, the way this is achieved within α-helical systems is different than in β-sheet or loop regions of proteins. These interactions between metal coordination preference and protein structural preference undoubtedly are exploited in natural systems to allow for protein conformation changes that define function. Thus, using a design approach that separates the numerous factors that lead to stable natural proteins allows us to extract fundamental concepts on how metals behave in biological systems.

  13. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...

  14. Monitoring the progression of calcium and protein solubilisation as affected by calcium chelators during small-scale manufacture of casein-based food matrices.

    Science.gov (United States)

    McIntyre, Irene; O'Sullivan, Michael; O'Riordan, Dolores

    2017-12-15

    Calcium and protein solubilisation during small-scale manufacture of semi-solid casein-based food matrices was investigated and found to be very different in the presence or absence of calcium chelating salts. Calcium concentrations in the dispersed phase increased and calcium-ion activity (ACa++) decreased during manufacture of the matrices containing calcium chelating salts; with ∼23% of total calcium solubilised by the end of manufacture. In the absence of calcium chelating salts, these concentrations were significantly lower at equivalent processing times and remained unchanged as did ACa++, throughout manufacture. The protein content of the dispersed phase was low (≤3% of total protein), but was significantly higher for matrices containing calcium chelating salts. This study elucidates the critical role of calcium chelating salts in modulating casein hydration and dispersion and gives an indication of the levels of soluble calcium and protein required to allow matrix formation during manufacture of casein-based food structures e.g. processed and analogue cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-06-01

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

  16. TAK1-binding protein 1 is a pseudophosphatase

    OpenAIRE

    Conner, Sarah H; Kular, Gursant; Peggie, Mark; Shepherd, Sharon; Schüttelkopf, Alexander W; Cohen, Philip; van Aalten, Daan M.F.

    2006-01-01

    Abstract TAB1 (TAK1-binding protein 1) is one of the regulatory subunits of TGF{beta}-activated kinase 1 (TAK1), a protein kinase that lies at the head of three pro-inflammatory kinase cascades. Here we report the crystal structure of the N-terminal domain of TAB1. Surprisingly, TAB1 possesses a fold closely related to that of the PPM phosphatase family as demonstrated by the close structural similarity with protein phosphatase 2C{alpha}. However, we were unable to detect any phosp...

  17. Engineering periplasmic ligand binding proteins as glucose nanosensors

    Directory of Open Access Journals (Sweden)

    Constance J. Jeffery

    2011-01-01

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

  18. New fluorescent probes for ligand-binding assays of odorant-binding proteins.

    Science.gov (United States)

    Mastrogiacomo, Rosa; Iovinella, Immacolata; Napolitano, Elio

    2014-03-28

    Fluorescence-linked binding assays allow determination of dissociation constants at equilibrium and have recently become increasingly popular, thanks to their ease of operation. Currently used probes, such as 1-aminoanthracene and N-phenyl-1-naphthylamine, are excited and emit in the ultraviolet region, but alternative ligands operating in the visible spectrum would be highly desirable for applications in biosensing devices. Based on the two above structures, we have designed and synthesised six new fluorescent probes to be used in ligand-binding assays. The compounds are derivatives of naphatalene, anthracene and fluoranthene and present two aromatic moieties linked by an amine nitrogen. We have measured the emission spectra of the new probes and their binding to three odorant-binding proteins. The probes bind the tested proteins with different affinities, generally with dissociation constants about one order of magnitude lower than the parent compounds. The extended aromatic systems present in the new compounds produced a shift of both excitation and emission peaks at higher wavelength, close or within the visible spectrum, thus facilitating measurements in biosensors for odorants and small organic molecules using optical devices. Copyright © 2014. Published by Elsevier Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-01

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

  20. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Science.gov (United States)

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

  1. Nicotine reward and affective nicotine withdrawal signs are attenuated in calcium/calmodulin-dependent protein kinase IV knockout mice.

    Directory of Open Access Journals (Sweden)

    Kia J Jackson

    Full Text Available The influx of Ca(2+ through calcium-permeable nicotinic acetylcholine receptors (nAChRs leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB, which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (-/- mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV -/- mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.

  2. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif) Protein to Heavy Metals

    OpenAIRE

    Kamala Boonyodying; Thanakorn Watcharasupat; Waranan Yotpanya; Thawatchai Kitti; Wanna Kawang; Duangkamol Kunthalert; Sutthirat Sitthisak

    2012-01-01

    A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif) recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to...

  3. Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole 'A' and calcium binding sites.

    Science.gov (United States)

    Ikeda, Minami; Kobayashi, Tamaki; Arai, Shinpei; Mukai, Saki; Takezawa, Yuka; Terasawa, Fumiko; Okumura, Nobuo

    2014-08-01

    We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio=0.295), suggesting hypodysfibrinogenemia. DNA sequence analysis was performed, and γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen. DNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1mM calcium ion compared with that of γN308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob 'A') and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole 'a' and D:D interaction sites were all markedly impaired, whereas γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole 'a'. Our findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2008-10-23

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-27

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

  6. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    Directory of Open Access Journals (Sweden)

    Grant S. Nichols

    2015-01-01

    Full Text Available Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX, the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults.

  7. Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

    Science.gov (United States)

    Puckett, Libby G; Dikici, Emre; Lai, Siyi; Madou, Marc; Bachas, Leonidas G; Daunert, Sylvia

    2004-12-15

    The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

  8. Characterization of a calcium-soluble protein fraction from yellow mustard (Sinapis alba) seed meal with potential application as an additive to calcium-rich drinks.

    Science.gov (United States)

    Aluko, Rotimi E; Reaney, Martin; McIntosh, Tara; Ouellet, François; Katepa-Mupondwa, Felicitas

    2004-09-22

    A calcium-soluble protein isolate (CSPI) was prepared from the supernatant obtained after addition of 0.75 M calcium chloride to a pH 5.0 aqueous extract of yellow mustard (Sinapis alba) seed meal. Total amino acid analysis showed that the CSPI has significantly higher (p proteins. Peptide mass fingerprinting of tryptic peptides of the major polypeptides by mass spectrometry indicated that the CSPI is composed mainly of cruciferin proteins with a contribution from napins (the major allergenic proteins of S. alba). The S. alba CSPI had significantly higher (p protein solubility and emulsion formation ability in the presence of 0.75 M calcium chloride when compared to similar isolates prepared from Brassica juncea (brown mustard) and soybean seed meals. We suggest that the S. alba CSPI could be used to prepare calcium-fortified high protein liquid products. However, the presence of allergenic proteins in this extract may limit its widespread food use.

  9. Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity.

    Science.gov (United States)

    Korkuć, Paula; Walther, Dirk

    2015-01-01

    To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous

  10. Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

    Science.gov (United States)

    Korkuć, Paula; Walther, Dirk

    2015-01-01

    To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous

  11. ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces *

    Science.gov (United States)

    Oroz, Javier; Barrera-Vilarmau, Susana; Alfonso, Carlos; Rivas, Germán; de Alba, Eva

    2016-01-01

    Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC·ASC and ASC·NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling. PMID:27432880

  12. Peeping into human renal calcium oxalate stone matrix: characterization of novel proteins involved in the intricate mechanism of urolithiasis.

    Directory of Open Access Journals (Sweden)

    Kanu Priya Aggarwal

    Full Text Available BACKGROUND: The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal-membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure. METHODS: Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin-Darby Canine Kidney (MDCK renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated. RESULTS: Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. CONCLUSIONS: We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone

  13. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

    Science.gov (United States)

    Sinha, Deepak K; Chandran, Predeesh; Timm, Alicia E; Aguirre-Rojas, Lina; Smith, C Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion.

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

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

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

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

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

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

  16. Binding of monoclonal antibody to CD16 causes calcium mobilization in large granular lymphocytes but inhibits NK killing.

    Science.gov (United States)

    Macintyre, E A; Wallace, D W; O'Flynn, K; Abdul-Gaffar, R; Tetteroo, P A; Morgan, G; Linch, D C

    1989-01-01

    A monoclonal antibody (mAb), CLB/FcR gran I, reactive with the CD16 Fc receptor (FcRlo/FcRIII) of human cells, leads to calcium mobilization in large granular lymphocytes (LGL) but not in granulocytes. Identical responses are obtained with F(ab')2 fragments of this antibody, indicating that the response is independent of Fc-FcR binding, and that bivalent cross-linking of this receptor is adequate for optimal calcium mobilization. The calcium response was greater in CD3- LGL compared to CD3+ LGL, although the response was augmented in the latter cells by prior rosetting with sheep red blood cells (SRBC). Calcium mobilization in CD3- LGL induced by CLB/FcR gran I is associated with inhibition of natural killer cell (NK) killing, and inhibition of the enhanced NK killing induced by the anti-CD2 low-density monoclonal antibody, 9.1. This supports the view that the NK-enhancing activity of 9.1 is due to simultaneous binding to CD2 and CD16, and may in fact be transduced through the CD16 molecule. The variable reported effects of anti-CD16 antibodies on NK killing are likely to reflect the epitope bound rather than the isotype of antibody used, since F(ab')2 fragments of CLB/FcR gran I also inhibit NK killing. PMID:2564843

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

    Science.gov (United States)

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

    2017-01-01

    Abstract The steroid hormone 17α‐hydroxylprogesterone (17‐OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17‐OHP containing an extended, nonpolar, shape‐complementary binding pocket for the four‐ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17‐OHP with micromolar affinity. A co‐crystal structure of one of the designs revealed that 17‐OHP is rotated 180° around a pseudo‐two‐fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same “flipped” orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two‐fold symmetry of the molecule. PMID:28980354

  18. Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

    Science.gov (United States)

    Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

    2007-04-13

    The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

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

    Science.gov (United States)

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

    2015-03-01

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

  20. [Liquid chromatography frontal analysis of the protein binding of glimepiride].

    Science.gov (United States)

    Zhou, Da-wei; Wang, Huai-feng; Li, Fa-mei

    2005-01-01

    To study the protein binding of glimepiride. An HPLC-FA method is performed by using Pinkerton GFF II-S5-80 internal-surface reversed-phase silica support (150 mm x 4.6 mm ID, 5 microm) at pH 7.4 in a 67 mmol x L(-1) isotonic sodium phosphate buffer at 37 degree C. Other conditions included flow rate of 0.2 mL x min(-1), UV detection at wavelength 230 nm and injection volume 900 microL. Nonlinear regression parameter estimation was used for the association constant measurement of glimepiride to both primary and secondary sites, which were 5.1 (micromol x L(-1)-1 and 1 for K1 and n1, and 0.017 (micromol x L(-1))-1 and 7 for K2 and n2, respectively. The method is shown to be suitable for investigation of protein binding of glimepiride.

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

    Science.gov (United States)

    Chen, Eileen; Joseph, Simpson

    2015-07-01

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

  2. Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα.

    Science.gov (United States)

    Fang, Changming; Filipp, Fabian V; Smith, Jeffrey W

    2012-04-01

    Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

  3. Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel.

    Science.gov (United States)

    Maturana, Andrés D; Wälchli, Sébastien; Iwata, Miki; Ryser, Stephan; Van Lint, Johannes; Hoshijima, Masahiko; Schlegel, Werner; Ikeda, Yasuhiro; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2008-06-01

    In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.

  4. QSAR Models for the Prediction of Plasma Protein Binding

    Directory of Open Access Journals (Sweden)

    Zeshan Amin

    2013-02-01

    Full Text Available Introduction: The prediction of plasma protein binding (ppb is of paramount importance in the pharmacokinetics characterization of drugs, as it causes significant changes in volume of distribution, clearance and drug half life. This study utilized Quantitative Structure – Activity Relationships (QSAR for the prediction of plasma protein binding. Methods: Protein binding values for 794 compounds were collated from literature. The data was partitioned into a training set of 662 compounds and an external validation set of 132 compounds. Physicochemical and molecular descriptors were calculated for each compound using ACD labs/logD, MOE (Chemical Computing Group and Symyx QSAR software packages. Several data mining tools were employed for the construction of models. These included stepwise regression analysis, Classification and Regression Trees (CART, Boosted trees and Random Forest. Results: Several predictive models were identified; however, one model in particular produced significantly superior prediction accuracy for the external validation set as measured using mean absolute error and correlation coefficient. The selected model was a boosted regression tree model which had the mean absolute error for training set of 13.25 and for validation set of 14.96. Conclusion: Plasma protein binding can be modeled using simple regression trees or multiple linear regressions with reasonable model accuracies. These interpretable models were able to identify the governing molecular factors for a high ppb that included hydrophobicity, van der Waals surface area parameters, and aromaticity. On the other hand, the more complicated ensemble method of boosted regression trees produced the most accurate ppb estimations for the external validation set.

  5. Effect of paracetamol on the plasma protein binding of quinine ...

    African Journals Online (AJOL)

    This study was done to investigate if the co-administration of quinine and paracetamol could have an effect on the protein binding of quinine. Exactly 5ml of plasma contained in a dialysis sack was placed into 20ml of varying concentrations (2 -10µg/ml) of test sample at 37oC. 2ml of the dialysate was basified with ...

  6. Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri.

    Science.gov (United States)

    Martínez-Castillo, Moisés; Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

    2015-01-01

    Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

  7. Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

    Directory of Open Access Journals (Sweden)

    Moisés Martínez-Castillo

    2015-01-01

    Full Text Available Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C employing conditioned medium (CM and total crude extracts (TCEs of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

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

    Science.gov (United States)

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

    1993-01-01

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

  9. Spatial Analysis and Quantification of the Thermodynamic Driving Forces in Protein-Ligand Binding: Binding Site Variability

    Science.gov (United States)

    Raman, E. Prabhu; MacKerell, Alexander D.

    2015-01-01

    The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  10. Folate and folate-binding protein content in dairy products.

    Science.gov (United States)

    Wigertz, K; Svensson, U K; Jägerstad, M

    1997-05-01

    Recent findings suggest a protective role for folates in the reduction of neural tube defects and possibly also coronary heart disease and cancer. Consequently, an increase in the daily intake of folates is warranted, which emphasizes the need for quantitative as well as qualitative measurements of dietary folates. Milk plays an important part in the food chain in many Western countries today. Several studies suggest that folate-binding proteins might have an impact on folate absorption and therefore their concentrations are also important. The mean concentration of the predominant form of folate, 5-methyltetrahydrofolate (5-CH3THF), was determined using HPLC in thirteen selected dairy products; skim milk powder, two pasteurized milks, UHT milk, two fermented milks, three whey products and four different cheeses. All results were corrected for recovery by spiking the samples with 5-CH3THF. Effects of storage of dairy products on 5-CH3THF concentrations were also investigated; generally small and insignificant fluctuations were found, except for hard cheese, in which 5-CH3THF decreased significantly. There was a significant seasonal variation in the folate concentration of pasteurized milk which peaked in the summer months. The concentrations of folate-binding protein in skim milk powder and pasteurized milk analysed using an enzyme-linked immunosorbent assay were similar. UHT milk and fermented milk, both of which are processed at temperatures > 90 degrees C, contained significantly lower concentrations of folate-binding protein.

  11. Phyletic distribution of fatty acid-binding protein genes.

    Directory of Open Access Journals (Sweden)

    Yadong Zheng

    Full Text Available Fatty acid-binding proteins (FABPs are a family of fatty acid-binding small proteins essential for lipid trafficking, energy storage and gene regulation. Although they have 20 to 70% amino acid sequence identity, these proteins share a conserved tertiary structure comprised of ten beta sheets and two alpha helixes. Availability of the complete genomes of 34 invertebrates, together with transcriptomes and ESTs, allowed us to systematically investigate the gene structure and alternative splicing of FABP genes over a wide range of phyla. Only in genomes of two cnidarian species could FABP genes not be identified. The genomic loci for FABP genes were diverse and their genomic structure varied. In particular, the intronless FABP genes, in most of which the key residues involved in fatty acid binding varied, were common in five phyla. Interestingly, several species including one trematode, one nematode and four arthropods generated FABP mRNA variants via alternative splicing. These results demonstrate that both gene duplication and post-transcriptional modifications are used to generate diverse FABPs in species studied.

  12. Enhanced expression of a calcium-dependent protein kinase from ...

    Indian Academy of Sciences (India)

    Unknown

    low calcium medium; LNM, low nitrate medium; LPM, low phosphate medium; LSM, low sulphate medium; MMG, minimal medium with glucose; NR, nitrate ... processes like pollen tube growth (Picton and Steer 1983) and cytokinin induced bud ... melanogaster and Saccharomyces cerevisiae. The CDPK genes are highly ...

  13. A short introduction to the new principle of binding ration calcium with sodium zeolite

    DEFF Research Database (Denmark)

    Jørgensen, R J; Bjerrum, M J; Classen, H

    2003-01-01

    This paper summarise the development of the new principle of preventing parturient hypocalcaemia by reducing the bioavailability of ration calcium with calcium binders, based on the idea that a negative calcium balance would stimulate natural defence mechanisms against threatening hypocalcaemia...... seen in these animals, the final proof of concept was done on pregnant dry cows fed a supplement of synthetic sodium zeolite A from 4 weeks before expected calving until calving. By analysis of blood calcium levels, this supplementation was shown to have a stabilizing effect during the critical period...

  14. The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

    Science.gov (United States)

    Pedrotti, Simona; Giudice, Jimena; Dagnino-Acosta, Adan; Knoblauch, Mark; Singh, Ravi K; Hanna, Amy; Mo, Qianxing; Hicks, John; Hamilton, Susan; Cooper, Thomas A

    2015-04-15

    The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein.

    Science.gov (United States)

    Kim, Juri; Nagami, Sara; Lee, Kyu-Ho; Park, Soon-Jung

    2014-01-01

    End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102-238, but not rGlEB11-184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11-238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

  16. Characterization of Microtubule-Binding and Dimerization Activity of Giardia lamblia End-Binding 1 Protein

    Science.gov (United States)

    Kim, Juri; Nagami, Sara; Lee, Kyu-Ho; Park, Soon-Jung

    2014-01-01

    End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102–238, but not rGlEB11–184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11–238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia. PMID:24828878

  17. Calcium excretion, apparent calcium absorption and calcium balance in young and elderly subjects: Influence of protein intake

    NARCIS (Netherlands)

    Pannemans, D.L.E.; Schaafsma, G.; Westerterp, K.R.

    1997-01-01

    The present study was conducted to investigate the effect of dietary protein on urinary Ca excretion, apparent Ca absorption and Ca balance in young and elderly subjects. Young adults (n 29) and elderly persons (n 26) consumed diets containing 12% (diet A) and 21% (diet B) of total energy as protein

  18. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose...

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    Science.gov (United States)

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

  1. Fatty acid- and retinoid-binding proteins have distinct binding pockets for the two types of cargo

    NARCIS (Netherlands)

    Jordanova, Rositsa; Groves, Matthew R.; Kostova, Elena; Woltersdorf, Christian; Liebau, Eva; Tucker, Paul A.

    2009-01-01

    Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described,

  2. A novel chitin binding crayfish molar tooth protein with elasticity properties.

    Directory of Open Access Journals (Sweden)

    Jenny Tynyakov

    Full Text Available The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate. This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.

  3. A novel chitin binding crayfish molar tooth protein with elasticity properties.

    Science.gov (United States)

    Tynyakov, Jenny; Bentov, Shmuel; Abehsera, Shai; Khalaila, Isam; Manor, Rivka; Katzir Abilevich, Lihie; Weil, Simy; Aflalo, Eliahu D; Sagi, Amir

    2015-01-01

    The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.

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

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

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

  5. Accesion number Protein name ENOA_MOUSE Alpha-enolase ...

    Indian Academy of Sciences (India)

    Sandra Feijoo Bandin

    Mitochondrial inner membrane protein. CMC1_MOUSE. Calcium-binding mitochondrial carrier protein Aralar1. CMC2_MOUSE. Calcium-binding mitochondrial carrier protein Aralar2. Biological process. Metabolic process. Glycolysis. Lipid metabolism. Respiratory electron transport chain. Others. Calcium ion homeostasis.

  6. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    Science.gov (United States)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  7. Regulation of Intestinal Epithelial Calcium Transport Proteins by Stanniocalcin-1 in Caco2 Cells

    Directory of Open Access Journals (Sweden)

    Jinmei Xiang

    2016-07-01

    Full Text Available Stanniocalcin-1 (STC1 is a calcium and phosphate regulatory hormone. However, the exact molecular mechanisms underlying how STC1 affects Ca2+ uptake remain unclear. Here, the expression levels of the calcium transport proteins involved in transcellular transport in Caco2 cells were examined following over-expression or inhibition of STC1. These proteins include the transient receptor potential vanilloid members (TRPV 5 and 6, the plasma membrane calcium ATPase 1b (PMCA1b, the sodium/calcium exchanger (NCX1, and the vitamin D receptor (VDR. Both gene and protein expressions of TRPV5 and TRPV6 were attenuated in response to over-expression of STC1, and the opposite trend was observed in cells treated with siRNASTC1. To further investigate the ability of STC1 to influence TRPV6 expression, cells were treated with 100 ng/mL of recombinant human STC1 (rhSTC1 for 4 h following pre-transfection with siRNASTC1 for 48 h. Intriguingly, the increase in the expression of TRPV6 resulting from siRNASTC1 was reversed by rhSTC1. No significant effect of STC1 on the expression of PMCA1b, NCX1 or VDR was observed in this study. In conclusion, the effect of STC1 on calcium transport in intestinal epithelia is due to, at least in part, its negative regulation of the epithelial channels TRPV5/6 that mediate calcium influx.

  8. Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

    Science.gov (United States)

    Antosiewicz, D. M.; Polisensky, D. H.; Braam, J.

    1995-01-01

    TCH3 is an Arabidopsis touch (TCH) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.

  9. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  10. Cloud Computing for Protein-Ligand Binding Site Comparison

    Directory of Open Access Journals (Sweden)

    Che-Lun Hung

    2013-01-01

    Full Text Available The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  11. Cloud Computing for Protein-Ligand Binding Site Comparison

    Science.gov (United States)

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  12. Collagen-binding proteins of Streptococcus mutans and related streptococci.

    Science.gov (United States)

    Avilés-Reyes, A; Miller, J H; Lemos, J A; Abranches, J

    2017-04-01

    The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. The telomere binding protein TRF2 induces chromatin compaction.

    Science.gov (United States)

    Baker, Asmaa M; Fu, Qiang; Hayward, William; Victoria, Samuel; Pedroso, Ilene M; Lindsay, Stuart M; Fletcher, Terace M

    2011-04-19

    Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  14. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

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

    Science.gov (United States)

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

    2016-11-01

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

  16. The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway*

    OpenAIRE

    Jing, Yu-Pu; Wang, Di; Han, Xiao-Lin; Dong, Du-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2016-01-01

    Animal steroid hormones regulate gene transcription through genomic pathways by binding to nuclear receptors. These steroid hormones also rapidly increase intracellular calcium and cyclic adenosine monophosphate (cAMP) levels and activate the protein kinase C (PKC) and protein kinase A (PKA) nongenomic pathways. However, the function and mechanism of the nongenomic pathways of the steroid hormones are unclear, and the relationship between the PKC and PKA pathways is also unclear. We propose t...

  17. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Dynamic competitive adsorption of bone-related proteins on calcium phosphate ceramic particles with different phase composition and microstructure.

    Science.gov (United States)

    Wang, Jing; Zhang, Huijie; Zhu, Xiangdong; Fan, Hongsong; Fan, Yujiang; Zhang, Xingdong

    2013-08-01

    The biocompatibility and bioactivity of biomaterials used for hard tissue repair are closely related to their adsorption capacities for bone-related proteins. In the present study, three types of calcium phosphate (CaP) ceramic particles with different phase composition or microstructure were fabricated, and their protein adsorption abilities were investigated by a self-made device under the simulated dynamic physiological circumstance. The results of X-ray diffraction, field emission scanning electron microscopy, mercury penetration test, and nitrogen sorption test showed that the irregular hydroxyapatite (HA) ceramic particles obtained by conventional drying and sintering (named as HA-C) had fewer micropores and lower specific surface area (SSA) than did the spherical HA or biphasic calcium phosphate (BCP) ceramic particles made by spray drying and sintering (named as HA-S and BCP-S, respectively). The dynamic protein adsorption study proved that both the phase composition and microstructure of CaP ceramic particles affected their adsorption capacities for those bone-related proteins. The spherical HA-S and BCP-S particles with abundant micropores and high SSA showed higher adsorption of serum proteins, including fibronectin and vitronectin, than the irregular HA-C did. On the other hand, in spite of the relatively high concentration of bovine serum albumin (BSA) in the binary bone morphogenetic protein 2 (BMP-2)/BSA solution, BMP-2 adsorption on the three CaP ceramic particles increased with the increase in its initial concentration. Similarly, HA-S and BCP-S particles had a larger amount of the adsorbed BMP-2 per gram solid than HA-C did. Therefore, it could be believed that the difference of various CaP ceramics in the phase composition and microporous structure would affect their binding capacity for those bone-related proteins and thus lead to their difference in osteoinduction. Copyright © 2013 Wiley Periodicals, Inc.

  2. Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

    Science.gov (United States)

    Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

    2017-06-21

    For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (CaVβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVβ interactions and reduce the entropic penalty associated with AID binding to CaVβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVβ interaction that modulate CaV function in an CaVβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design.

  3. The Movable Type Method Applied to Protein-Ligand Binding.

    Science.gov (United States)

    Zheng, Zheng; Ucisik, Melek N; Merz, Kenneth M

    2013-12-10

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term "movable type". Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the free

  4. Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs.

    Directory of Open Access Journals (Sweden)

    Ramakrishnan B Kumar

    Full Text Available An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1 produce the expected leukotriene products from arachidonic acid and 2 can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3 show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

  5. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  6. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  7. Control of nuclear organization by F-actin binding proteins.

    Science.gov (United States)

    Pfisterer, Karin; Jayo, Asier; Parsons, Maddy

    2017-03-04

    The regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

  8. Rbfox2 controls autoregulation in RNA-binding protein networks.

    Science.gov (United States)

    Jangi, Mohini; Boutz, Paul L; Paul, Prakriti; Sharp, Phillip A

    2014-03-15

    The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

  9. Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

    Science.gov (United States)

    Lu, Y. T.; Hidaka, H.; Feldman, L. J.

    1996-01-01

    Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

  10. Proteomic identification of calcium-binding chaperone calreticulin as a potential mediator for the neuroprotective and neuritogenic activities of fruit-derived glycoside amygdalin.

    Science.gov (United States)

    Cheng, Yuanyuan; Yang, Chuanbin; Zhao, Jia; Tse, Hung Fat; Rong, Jianhui

    2015-02-01

    Amygdalin is a fruit-derived glycoside with the potential for treating neurodegenerative diseases. This study was designed to identify the neuroprotective and neuritogenic activities of amygdalin. We initially demonstrated that amygdalin enhanced nerve growth factor (NGF)-induced neuritogenesis and attenuated 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in rat dopaminergic PC12 cells. To define protein targets for amygdalin, we selected a total of 11 mostly regulated protein spots from two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels for protein identification by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry. We verified the effect of amygdalin on six representative proteins (i.e., calreticulin, Hsp90β, Grp94, 14-3-3η, 14-3-3ζ/δ and Rab GDI-α) for biological relevance to neuronal survival and differentiation. Calcium-binding chaperone calreticulin is of special interest for its activities to promote folding, oligomeric assembly and quality control of proteins that modulate cell survival and differentiation. We transiently knocked down calreticulin expression by specific siRNA and studied its effect on the neuroprotective and neuritogenic activities of amygdalin. We found that amygdalin failed to enhance NGF-induced neuritogenesis in calreticulin-siRNA transfected cells. On the other hand, amygdalin rescued 6-OHDA-induced loss of calreticulin expression. We also found that amygdalin increased the intracellular calcium concentration possibly via inducing calreticulin. Collectively, our results demonstrated the role of calreticulin in mediating the neuroprotective and neuritogenic activities of amygdalin. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

    Science.gov (United States)

    van der Vaart, Arjan

    2015-05-01

    Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

  12. Conjugation Dependent Interaction of Folic Acid with Folate Binding Protein.

    Science.gov (United States)

    Merzel, Rachel L; Frey, Carolina; Chen, Junjie; Garn, Rachel; van Dongen, Mallory; Dougherty, Casey A; Kandaluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

    2017-09-20

    Serum proteins play a critical role in the transport, uptake, and efficacy of targeted drug therapies, and here we investigate the interactions between folic acid-polymer conjugates and serum folate binding protein (FBP), the soluble form of the cellular membrane-bound folate receptor. We demonstrate that both choice of polymer and method of ligand conjugation affect the interactions between folic acid-polymer conjugates and serum FBP, resulting in changes in the folic acid-induced protein aggregation process. We have previously demonstrated that individual FBP molecules self-aggregate into nanoparticles at physiological concentrations. When poly(amidoamine) dendrimer-folic acid conjugates bound to FBP, the distribution of nanoparticles was preserved. However, the dendritic conjugates produced larger nanoparticles than those formed in the presence of physiologically normal human levels of folic acid, and the conjugation method affected particle size distribution. In contrast, poly(ethylene glycol)-folic acid conjugates demonstrated substantially reduced binding to FBP, did not cause folic acid-induced aggregation, and fully disrupted FBP self-aggregation. On the basis of these results, we discuss the potential implications for biodistribution, trafficking, and therapeutic efficacy of targeted nanoscale therapeutics, especially considering the widespread clinical use of poly(ethylene glycol) conjugates. We highlight the importance of considering specific serum protein interactions in the rational design of similar nanocarrier systems. Our results suggest that prebinding therapeutic nanocarriers to serum FBP may allow folate-specific metabolic pathways to be exploited for delivery while also affording benefits of utilizing an endogenous protein as a vector.

  13. Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

    Directory of Open Access Journals (Sweden)

    Seon-Hee Kim

    Full Text Available BACKGROUND: Fatty acid (FA binding proteins (FABPs of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs of Taenia solium metacestode (TsM, a causative agent of neurocysticercosis (NC, shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2, which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1 and 8.4 (TsMFABP2. Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]aminoundecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions

  14. Serum lipopolysaccharide-binding protein as a marker of atherosclerosis.

    Science.gov (United States)

    Serrano, Marta; Moreno-Navarrete, José María; Puig, Josep; Moreno, María; Guerra, Ester; Ortega, Francisco; Xifra, Gemma; Ricart, Wifredo; Fernández-Real, José Manuel

    2013-10-01

    Recently, serum lipopolysaccharide-binding protein (LBP) has been closely associated with coronary artery disease. Here, we aimed to investigate the possible relationship between serum LBP and markers of atherosclerosis. Serum LBP and carotid intima media thickness (C-IMT) were measured in 332 subjects (101 men and 231 women) who were recruited from an ongoing multicenter project. Serum LBP was significantly associated with obesity [BMI, fat mass and waist circumference (r > 0.38, p atherosclerosis marker, reveals serum LBP as a putative factor related to atherosclerosis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also foun...

  16. Prediction of RNA-Binding Proteins by Voting Systems

    Directory of Open Access Journals (Sweden)

    C. R. Peng

    2011-01-01

    Full Text Available It is important to identify which proteins can interact with RNA for the purpose of protein annotation, since interactions between RNA and proteins influence the structure of the ribosome and play important roles in gene expression. This paper tries to identify proteins that can interact with RNA using voting systems. Firstly through Weka, 34 learning algorithms are chosen for investigation. Then simple majority voting system (SMVS is used for the prediction of RNA-binding proteins, achieving average ACC (overall prediction accuracy value of 79.72% and MCC (Matthew’s correlation coefficient value of 59.77% for the independent testing dataset. Then mRMR (minimum redundancy maximum relevance strategy is used, which is transferred into algorithm selection. In addition, the MCC value of each classifier is assigned to be the weight of the classifier’s vote. As a result, best average MCC values are attained when 22 algorithms are selected and integrated through weighted votes, which are 64.70% for the independent testing dataset, and ACC value is 82.04% at this moment.

  17. Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Nielsen, EH; Andersen, Ove

    1996-01-01

    Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy....... Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high...... affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate...

  18. Calcium, Copper Protein And Oxygen Affinity In Haemocyanins Of ...

    African Journals Online (AJOL)

    ... to buffer the decrease in extracellular pH during aestivation is likely responsible for the high oxygen affinity of haemocyanin (43.0% increase) from aestivating snails through co-operative oxygen binding. Key Words: Aestivation, snail, Achatina achatina, inorganic ions, haemocyanin, absorption spectra, oxygen affinity.

  19. Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter.

    Science.gov (United States)

    Cao, Chan; Wang, Shuqing; Cui, Tanxing; Su, Xun-Cheng; Chou, James J

    2017-04-04

    The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.

  20. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  1. The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

    DEFF Research Database (Denmark)

    Schrøder, Jacob Morville

    centrosomal MT array and abnormally long centriole-associated rootlet filaments. Cells lacking EB1 also had stumpy cilia and a disorganized centrosomal MT array, but rootlet filaments appeared normal. Further, live imaging revealed increased release frequency of MTs from the centrosome upon EB1 or EB3......EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends. EB1 plays a role in regulating MT dynamics, localizing other MT-associated proteins to the plus end, and in regulating interactions of MTs with the cell cortex, mitotic kinetochores and different......, are required for assembly of primary cilia in cultured human cells. The EB3 - siRNA ciliary phenotype could be rescued by GFP-EB1 expression, and GFP-EB3 over expression resulted in elongated cilia. Transmission electron microscopy (TEM) revealed that EB3-depleted cells possess stumpy cilia, a disorganized...

  2. Mapping protein binding sites on the biomolecular corona of nanoparticles

    Science.gov (United States)

    Kelly, Philip M.; Åberg, Christoffer; Polo, Ester; O'Connell, Ann; Cookman, Jennifer; Fallon, Jonathan; Krpetić, Željka; Dawson, Kenneth A.

    2015-05-01

    Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized ‘corona’ of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.

  3. Comparison of entropic contributions to binding in a "hydrophilic" versus "hydrophobic" ligand-protein interaction.

    Science.gov (United States)

    Syme, Neil R; Dennis, Caitriona; Bronowska, Agnieszka; Paesen, Guido C; Homans, Steve W

    2010-06-30

    In the present study we characterize the thermodynamics of binding of histamine to recombinant histamine-binding protein (rRaHBP2), a member of the lipocalin family isolated from the brown-ear tick Rhipicephalus appendiculatus. The binding pocket of this protein contains a number of charged residues, consistent with histamine binding, and is thus a typical example of a "hydrophilic" binder. In contrast, a second member of the lipocalin family, the recombinant major urinary protein (rMUP), binds small hydrophobic ligands, with a similar overall entropy of binding in comparison with rRaHBP2. Having extensively studied ligand binding thermodynamics for rMUP previously, the data we obtained in the present study for HBP enables a comparison of the driving forces for binding between these classically distinct binding processes in terms of entropic contributions from ligand, protein, and solvent. In the case of rRaHBP2, we find favorable entropic contributions to binding from desolvation of the ligand; however, the overall entropy of binding is unfavorable due to a dominant unfavorable contribution arising from the loss of ligand degrees of freedom, together with the sequestration of solvent water molecules into the binding pocket in the complex. This contrasts with binding in rMUP where desolvation of the protein binding pocket makes a minor contribution to the overall entropy of binding given that the pocket is substantially desolvated prior to binding.

  4. Immobilized sialyloligo-macroligand and its protein binding specificity.

    Science.gov (United States)

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

    We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

  5. Protein-protein binding before and after photo-modification of albumin

    Science.gov (United States)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  6. Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography.

    Science.gov (United States)

    Cabanne, Charlotte; Pezzini, Jérôme; Joucla, Gilles; Hocquellet, Agnès; Barbot, Caroline; Garbay, Bertrand; Santarelli, Xavier

    2009-05-15

    Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.

  7. DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein.

    Science.gov (United States)

    Morin, José A; Cerrón, Fernando; Jarillo, Javier; Beltran-Heredia, Elena; Ciesielski, Grzegorz L; Arias-Gonzalez, J Ricardo; Kaguni, Laurie S; Cao, Francisco J; Ibarra, Borja

    2017-07-07

    Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  9. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  10. Activated macrophage survival is coordinated by TAK1 binding proteins.

    Directory of Open Access Journals (Sweden)

    September R Mihaly

    Full Text Available Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFβ- activated kinase (TAK1 activators, TAK1-binding protein 1 (TAB1 and TAK1-binding protein 2 (TAB2, are critical molecules in the regulation of activated macrophage survival. While deletion of Tak1 induced cell death in bone marrow derived macrophages even without activation, Tab1 or Tab2 deletion alone did not profoundly affect survival of naïve macrophages. However, in lipopolysaccharide (LPS-activated macrophages, even single deletion of Tab1 or Tab2 resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an in vivo setting, Tab1 deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in in vivo macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.

  11. Melanocortin receptor binding determinants in the agouti protein.

    Science.gov (United States)

    Kiefer, L L; Veal, J M; Mountjoy, K G; Wilkison, W O

    1998-01-27

    The agouti protein plays an important role in the development of diabetes and obesity in rodents and has been shown to be a potent antagonist of melanocortin receptors. For this reason alanine-scanning mutagenesis was performed on the agouti protein carboxyl terminus to locate residues important for melanocortin receptor binding inhibition. When agouti residues Arg116 and Phe118 are changed to alanine, very large decreases in agouti affinity for melanocortin receptor 1, 3, and 4 result. Mutation of Phe117 to alanine causes a similar increase in agouti KI app at melanocortin receptor 4. Substitution of agouti residue Asp108 with alanine results in large increases in KI app for all three melanocortin receptors examined. All of these residues are conserved in the agouti-related transcript, ART, whose expression is up-regulated in animal models of obesity. The three-dimensional structure of the agouti carboxyl terminus was modeled, and residues which decrease receptor binding by a factor of > or = 15 when mutated to alanine localize to one side of the structure. These agouti variants with altered receptor selectivity may be useful in determining the role of melanocortin receptors in diabetes and obesity.

  12. Simulation of Reversible Protein–Protein Binding and Calculation of Binding Free Energies Using Perturbed Distance Restraints

    Science.gov (United States)

    2017-01-01

    Virtually all biological processes depend on the interaction between proteins at some point. The correct prediction of biomolecular binding free-energies has many interesting applications in both basic and applied pharmaceutical research. While recent advances in the field of molecular dynamics (MD) simulations have proven the feasibility of the calculation of protein–protein binding free energies, the large conformational freedom of proteins and complex free energy landscapes of binding processes make such calculations a difficult task. Moreover, convergence and reversibility of resulting free-energy values remain poorly described. In this work, an easy-to-use, yet robust approach for the calculation of standard-state protein–protein binding free energies using perturbed distance restraints is described. In the binding process the conformations of the proteins were restrained, as suggested earlier. Two approaches to avoid end-state problems upon release of the conformational restraints were compared. The method was evaluated by practical application to a small model complex of ubiquitin and the very flexible ubiquitin-binding domain of human DNA polymerase ι (UBM2). All computed free energy differences were closely monitored for convergence, and the calculated binding free energies had a mean unsigned deviation of only 1.4 or 2.5 kJ·mol–1 from experimental values. Statistical error estimates were in the order of thermal noise. We conclude that the presented method has promising potential for broad applicability to quantitatively describe protein–protein and various other kinds of complex formation. PMID:28898077

  13. The telomere binding protein TRF2 induces chromatin compaction.

    Directory of Open Access Journals (Sweden)

    Asmaa M Baker

    2011-04-01

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  14. Ionized calcium-binding adaptor molecule 1 positive macrophages and HO-1 up-regulation in intestinal muscularis resident macrophages

    DEFF Research Database (Denmark)

    Mikkelsen, Hanne B; Huizinga, Jan D; Larsen, Jytte O

    2017-01-01

    the reaction of resident macrophages of the musculature to a pro-inflammatory stimulator, lipopolysaccharide (LPS). Mice were injected with LPS or saline and sacrificed after 6 hours. Whole mounts were stained with antibodies toward CD169, ionized calcium-binding adaptor molecule 1 (iba1) (microglial...... macrophages in serosa and at AP, suggesting a M2 phenotype. LPS-treatment results in an up-regulation of HO-1(pos) /CD169(neg) cells in serosa and at AP. This article is protected by copyright. All rights reserved....

  15. Cloning and characterization of a calcium dependent protein kinase gene associated with cotton fiber development.

    Science.gov (United States)

    Huang, Quan-Sheng; Wang, Hai-Yun; Gao, Peng; Wang, Guo-Ying; Xia, Gui-Xian

    2008-12-01

    The gene GhCPK1 encoding a calcium dependent protein kinase was identified from cotton. Transcripts of GhCPK1 accumulated primarily in the elongating fiber, and Arabidopsis plants transformed with GhCPK1 promoter-GUS construct exhibited GUS activity mainly in the developing trichomes, roots, young leaves and sepals. In the bombarded onion epidermal cells, GhCPK1-GFP fusion proteins showed a subcellular distribution in the plasma membrane. In vitro assays indicated that GhCPK1 was a functional calcium-dependent kinase able to undergo autophosphorylation and phosphorylation of the known substrate histone III-S. Together, these results suggest that GhCPK1 may play a role in the calcium signaling events associated with fiber elongation.

  16. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  17. Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants.

    Science.gov (United States)

    Ludwig, Andrea A; Saitoh, Hiromasa; Felix, Georg; Freymark, Gerald; Miersch, Otto; Wasternack, Claus; Boller, Thomas; Jones, Jonathan D G; Romeis, Tina

    2005-07-26

    Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress.

  18. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    Science.gov (United States)

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

  19. The function of DNA binding protein nucleophosmin in AAV replication.

    Science.gov (United States)

    Satkunanathan, Stifani; Thorpe, Robin; Zhao, Yuan

    2017-10-01

    Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  20. Direct protein-protein interactions and substrate channelling between cellular retinoic acid binding proteins and CYP26B1

    Science.gov (United States)

    Nelson, Cara H; Peng, Chi-Chi; Lutz, Justin D.; Yeung, Catherine K.; Zelter, Alex; Isoherranen, Nina

    2016-01-01

    Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates. PMID:27416800

  1. Novel approach for selecting the best predictor for identifying the binding sites in DNA binding proteins.

    Science.gov (United States)

    Nagarajan, R; Ahmad, Shandar; Gromiha, M Michael

    2013-09-01

    Protein-DNA complexes play vital roles in many cellular processes by the interactions of amino acids with DNA. Several computational methods have been developed for predicting the interacting residues in DNA-binding proteins using sequence and/or structural information. These methods showed different levels of accuracies, which may depend on the choice of data sets used in training, the feature sets selected for developing a predictive model, the ability of the models to capture information useful for prediction or a combination of these factors. In many cases, different methods are likely to produce similar results, whereas in others, the predictors may return contradictory predictions. In this situation, a priori estimates of prediction performance applicable to the system being investigated would be helpful for biologists to choose the best method for designing their experiments. In this work, we have constructed unbiased, stringent and diverse data sets for DNA-binding proteins based on various biologically relevant considerations: (i) seven structural classes, (ii) 86 folds, (iii) 106 superfamilies, (iv) 194 families, (v) 15 binding motifs, (vi) single/double-stranded DNA, (vii) DNA conformation (A, B, Z, etc.), (viii) three functions and (ix) disordered regions. These data sets were culled as non-redundant with sequence identities of 25 and 40% and used to evaluate the performance of 11 different methods in which online services or standalone programs are available. We observed that the best performing methods for each of the data sets showed significant biases toward the data sets selected for their benchmark. Our analysis revealed important data set features, which could be used to estimate these context-specific biases and hence suggest the best method to be used for a given problem. We have developed a web server, which considers these features on demand and displays the best method that the investigator should use. The web server is freely available at

  2. Comparative sperm protein profiling in bulls differing in fertility and identification of phosphatidylethanolamine-binding protein 4, a potential fertility marker.

    Science.gov (United States)

    Somashekar, L; Selvaraju, S; Parthipan, S; Patil, S K; Binsila, B K; Venkataswamy, M M; Karthik Bhat, S; Ravindra, J P

    2017-09-01

    This study aimed to identify sperm proteomic signatures regulating sperm functions and fertility by: (i) comparing the sperm electrophoretic protein profiles and identifying the differentially abundant proteins among breeding bulls differing in fertility status and (ii) elucidating the possible role of one of the identified novel proteins, PEBP4 on sperm function and fertility. The grouping of bulls as fertile (n = 6) and low fertile (n = 6) was performed based on bull fertility index and infertile (n = 6) based on semen rejection rate (>33%). The sperm motility, fructolysis index, acrosomal reaction, intracellular calcium levels, and seminal plasma fructose and calcium levels were studied among fertility groups. The differentially expressed sperm proteins observed in single- and two-dimensional gel electrophoresis (2DE) were identified using Nano-LC-MS/MS. In the fertile bulls, the expression levels of calmodulin (CALM1), spermadhesinZ13 (SPADH2), and phosphatidylethanolamine-binding protein 4 (PEBP4) were significantly (p sperm fructose uptake was observed. Further, PEBP4 was localized in elongated spermatids, Leydig cells, excurrent duct system, and principal piece of spermatozoa. These findings suggest a crucial role for the PEBP4 protein in spermiogenesis, epididymal sperm maturation, and sperm motility. This first study in bovine indicates the positive association of PEBP4 in regulating sperm maturation, functions, and fertility and could be a potential marker for predicting semen quality and fertility. © 2017 American Society of Andrology and European Academy of Andrology.

  3. Binding site prediction for protein-protein interactions and novel motif discovery using re-occurring polypeptide sequences.

    Science.gov (United States)

    Amos-Binks, Adam; Patulea, Catalin; Pitre, Sylvain; Schoenrock, Andrew; Gui, Yuan; Green, James R; Golshani, Ashkan; Dehne, Frank

    2011-06-02

    While there are many methods for predicting protein-protein interaction, very few can determine the specific site of interaction on each protein. Characterization of the specific sequence regions mediating interaction (binding sites) is crucial for an understanding of cellular pathways. Experimental methods often report false binding sites due to experimental limitations, while computational methods tend to require data which is not available at the proteome-scale. Here we present PIPE-Sites, a novel method of protein specific binding site prediction based on pairs of re-occurring polypeptide sequences, which have been previously shown to accurately predict protein-protein interactions. PIPE-Sites operates at high specificity and requires only the sequences of query proteins and a database of known binary interactions with no binding site data, making it applicable to binding site prediction at the proteome-scale. PIPE-Sites was evaluated using a dataset of 265 yeast and 423 human interacting proteins pairs with experimentally-determined binding sites. We found that PIPE-Sites predictions were closer to the confirmed binding site than those of two existing binding site prediction methods based on domain-domain interactions, when applied to the same dataset. Finally, we applied PIPE-Sites to two datasets of 2347 yeast and 14,438 human novel interacting protein pairs predicted to interact with high confidence. An analysis of the predicted interaction sites revealed a number of protein subsequences which are highly re-occurring in binding sites and which may represent novel binding motifs. PIPE-Sites is an accurate method for predicting protein binding sites and is applicable to the proteome-scale. Thus, PIPE-Sites could be useful for exhaustive analysis of protein binding patterns in whole proteomes as well as discovery of novel binding motifs. PIPE-Sites is available online at http://pipe-sites.cgmlab.org/.

  4. OB protein binds specifically to the choroid plexus of mice and rats.

    Science.gov (United States)

    Devos, R; Richards, J G; Campfield, L A; Tartaglia, L A; Guisez, Y; van der Heyden, J; Travernier, J; Plaetinck, G; Burn, P

    1996-05-28

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor.

  5. Inhibition of calcium/calmodulin-dependent protein kinase kinase β and calcium/calmodulin-dependent protein kinase IV is detrimental in cerebral ischemia.

    Science.gov (United States)

    McCullough, Louise D; Tarabishy, Sami; Liu, Lin; Benashski, Sharon; Xu, Yan; Ribar, Thomas; Means, Anthony; Li, Jun

    2013-09-01

    Elevation of intracellular calcium was traditionally thought to be detrimental in stroke pathology. However, clinical trials testing treatments that block calcium signaling have failed to improve outcomes in ischemic stroke. Emerging data suggest that calcium may also trigger endogenous protective pathways after stroke. Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is a major kinase activated by rising intracellular calcium. Compelling evidence has suggested that CaMKK and its downstream kinase CaMK IV are critical in neuronal survival when cells are under ischemic stress. We examined the functional role of CaMKK/CaMK IV signaling in stroke. We used a middle cerebral artery occlusion model in mice. Our data demonstrated that pharmacological and genetic inhibition of CaMKK aggravated stroke injury. Additionally, deletion of CaMKK β, one of the 2 CaMKK isoforms, reduced CaMK IV activation, and CaMK IV deletion in mice worsened stroke outcome. Finally, CaMKK β or CaMK IV knockout mice had exacerbated blood-brain barrier disruption evidenced by increased hemorrhagic transformation and activation of matrix metalloproteinase. We observed transcriptional inactivation including reduced levels of histone deacetylase 4 phosphorylation in mice with CaMKK β or CaMK IV deletion after stroke. Our data have established that the CaMKK/CaMK IV pathway is a key endogenous protective mechanism in ischemia. Our results suggest that this pathway serves as an important regulator of blood-brain barrier integrity and transcriptional activation of neuroprotective molecules in stroke.

  6. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

  7. Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit.

    Science.gov (United States)

    Herington, A C; Ymer, S; Roupas, P; Stevenson, J

    1986-04-11

    Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.

  8. CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation.

    Science.gov (United States)

    Pereira, Renata C; Delany, Anne M; Canalis, Ernesto

    2004-04-01

    CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP/DDIT3), a member of the C/EBP family of transcription factors, plays a role in cell survival and differentiation. CHOP/DDIT3 binds to C/EBPs to form heterodimers that do not bind to consensus Cebp sequences, acting as a dominant-negative inhibitor. CHOP/DDIT3 blocks adipogenesis, and we postulated it could induce osteoblastogenesis. We investigated the effects of constitutive CHOP/DDIT3 overexpression in murine ST-2 stromal cells transduced with retroviral vectors. ST-2 cells differentiated toward osteoblasts, and CHOP/DDIT3 accelerated and enhanced the appearance of mineralized nodules, and the expression of osteocalcin and alkaline phosphatase mRNAs, particularly in the presence of bone morphogenetic protein-2. CHOP/DDIT3 overexpression opposed adipogenesis, and did not cause substantial changes in cell number. CHOP/DDIT3 overexpression did not modify C/EBPalpha or -beta mRNA levels but decreased C/EBPdelta after 24 d of culture. Electrophoretic mobility shift and supershift assays demonstrated that overexpression of CHOP/DDIT3 decreased the binding of C/EBPs to their consensus sequence by interacting with C/EBPalpha and -beta, confirming its dominant-negative role. In addition, CHOP/DDIT3 enhanced bone morphogenetic protein-2/Smad signaling. In conclusion, CHOP/DDIT3 enhances osteoblastic differentiation of stromal cells, in part by interacting with C/EBPalpha and -beta and also by enhancing Smad signaling.

  9. Interacting protein partners of Arabidopsis RNA binding protein AtRBP45b

    Science.gov (United States)

    RNA binding proteins (RBPs) are important players in post-transcriptional gene regulation and shown to play an important role in normal development and in response to environmental perturbations. Arabidopsis RBP, AtRBP45b with triple RNA recognition motifs (RRMs) have are closely related to the yeas...

  10. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show that de...

  11. Down modulation of HIV-1 gene expression using a procaryotic RNA-binding protein

    NARCIS (Netherlands)

    Berkhout, B.; Jeang, K. T.

    1990-01-01

    The coat protein of the single stranded RNA bacteriophages acts as a translational repressor by binding with high affinity to a target RNA that encompasses the ribosomal binding site of the replicase gene. We have expressed this procaryotic RNA-binding protein in mammalian cells. Using the coat

  12. A single rainbow trout cobalamin-binding protein stands in for three human binders

    DEFF Research Database (Denmark)

    Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S

    2012-01-01

    -binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases...... affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation...

  13. Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII).

    Science.gov (United States)

    Zhu-Salzman, Keyan; Hammen, Philip K; Salzman, Ron A; Koiwa, Hisashi; Bressan, Ray A; Murdock, Larry L; Hasegawa, Paul M

    2002-06-01

    Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123-15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it dysfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca(2+) content. Accordingly, the re-addition of Ca(2+) to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca(2+)-dependent.

  14. Value of heart-type fatty acid-binding protein (H-FABP) for ...

    African Journals Online (AJOL)

    Key Words: heart-type fatty acid-binding protein, acute coronary syndrome, biomarker. DOI: http://dx.doi.org/10.4314/ahs.v14i3.36 ... weight (14.5 kDa) protein, which contains 132 amino acid residues (3). Fatty acid-binding proteins ..... injury by assessment of the plasma ratio of my- oglobin over fatty acid binding protein.

  15. Zn2+Interaction with Alzheimer Amyloid β Protein Calcium Channels

    National Research Council Canada - National Science Library

    Nelson Arispe; Harvey B. Pollard; Eduardo Rojas

    1996-01-01

    The Alzheimer disease 40-residue amyloid β protein (Aβ P[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS...

  16. Long-term cell-mediated protein release from calcium phosphate ceramics

    NARCIS (Netherlands)

    Wernike, E.; Hofstetter, W.; Liu, Y.; Wu, G.; Sebald, H.J.; Wismeijer, D.; Hunziker, E.B.; Siebenrock, K.A.; Klenke, F.M.

    2010-01-01

    Efficient delivery of growth factors from carrier biomaterials depends critically on the release kinetics of the proteins that constitute the carrier. Immobilizing growth factors to calcium phosphate ceramics has been attempted by direct adsorption and usually resulted in a rapid and passive release

  17. Animal products, calcium and protein and prostate cancer risk in the Netherlands Cohort Study

    NARCIS (Netherlands)

    Schuurman, A.G.; Brandt, P.A. van den; Dorant, E.; Goldbohm, R.A.

    1999-01-01

    Prostate cancer risk in relation to consumption of animal products, and intake of calcium and protein was investigated in the Netherlands Cohort Study. At baseline in 1986, 58,279 men aged 55-69 years completed a self-administered 150-item food frequency questionnaire and a questionnaire on other

  18. The aging human cochlear nucleus: Changes in the glial fibrillary acidic protein, intracellular calcium regulatory proteins, GABA neurotransmitter and cholinergic receptor.

    Science.gov (United States)

    Sharma, Saroj; Nag, Tapas C; Thakar, Alok; Bhardwaj, Daya N; Roy, Tara Sankar

    2014-03-01

    The human auditory system is highly susceptible to environmental and metabolic insults which further affect the biochemical and physiological milieu of the cells that may contribute to progressive, hearing loss with aging. The cochlear nucleus (CN) is populated by morphologically diverse types of neurons with discrete physiological and neurochemical properties. Between the dorsal and the ventral cochlear nucleus (DCN and VCN), the VCN is further sub-divided into the rostral (rVCN) and caudal (cVCN) sub-divisions. Although, information is available on the age related neurochemical changes in the mammalian CN similar reports on human CN is still sparse. The morphometry and semiquantitative analysis of intensity of expression of glial fibrillary acidic protein (GFAP), calcium binding proteins (calbindin, calretinin and parvalbumin), gamma amino butyric acid (GABA) and nicotinic acetyl choline receptor (nAchR) beta 2 immunostaining were carried out in all three sub-divisions of the human CN from birth to 90 years. There was increased GFAP immunoreactivity in decades 2 and 3 in comparison to decade 1 in the CN. But no change was observed in rVCN from decade 4 onwards, whereas intense staining was also observed in decades 5 and 6 in cVCN and DCN. All three calcium binding proteins were highly expressed in early to middle ages, whereas a significant reduction was found in later decades in the VCN. GABA and nAchR beta 2 expressions were unchanged throughout in all the decades. The middle age may represent a critical period of onset and progression of aging changes in the CN and these alterations may add to the deterioration of hearing responses in the old age. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Microfibril-associated Protein 4 Binds to Surfactant Protein A (SP-A) and Colocalizes with SP-A in the Extracellular Matrix of the Lung

    DEFF Research Database (Denmark)

    Schlosser, Anders; Thomsen, Theresa H.; Shipley, J. Michael

    2006-01-01

    Pulmonary surfactant protein A (SP-A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro-organisms, and mediates microbial lysis and clearance. SP-A also modulates multiple immune-related functions including cytokine production and chemotaxis...... for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4...... with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium-dependent binding between MFAP4 and human SP-A1 and SP-A2. No binding was seen to recombinant SP-A...

  20. MARCKS Protein Is Phosphorylated and Regulates Calcium Mobilization during Human Acrosomal Exocytosis

    Science.gov (United States)

    Rodriguez Peña, Marcelo J.; Castillo Bennett, Jimena V.; Soler, Osvaldo M.; Mayorga, Luis S.; Michaut, Marcela A.

    2013-01-01

    Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering

  1. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

    Directory of Open Access Journals (Sweden)

    Marcin Olszewski

    Full Text Available SSB (single-stranded DNA-binding proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein. This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity. The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m is 100.2 °C as shown by differential scanning calorimetry (DSC analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

  2. Accuracy of protein-protein binding sites in high-throughput template-based modeling.

    Directory of Open Access Journals (Sweden)

    Petras J Kundrotas

    2010-04-01

    Full Text Available The accuracy of protein structures, particularly their binding sites, is essential for the success of modeling protein complexes. Computationally inexpensive methodology is required for genome-wide modeling of such structures. For systematic evaluation of potential accuracy in high-throughput modeling of binding sites, a statistical analysis of target-template sequence alignments was performed for a representative set of protein complexes. For most of the complexes, alignments containing all residues of the interface were found. The full interface alignments were obtained even in the case of poor alignments where a relatively small part of the target sequence (as low as 40% aligned to the template sequence, with a low overall alignment identity (<30%. Although such poor overall alignments might be considered inadequate for modeling of whole proteins, the alignment of the interfaces was strong enough for docking. In the set of homology models built on these alignments, one third of those ranked 1 by a simple sequence identity criteria had RMSD<5 A, the accuracy suitable for low-resolution template free docking. Such models corresponded to multi-domain target proteins, whereas for single-domain proteins the best models had 5 Aprotein-protein complexes.

  3. The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp

    Directory of Open Access Journals (Sweden)

    Lei Benfang

    2008-01-01

    Full Text Available Abstract Background The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established. Results The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp. Conclusion We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.

  4. Application of immunoglobulin-binding proteins a, g, l in the affinity chromatography

    OpenAIRE

    Sviatenko, О.; Gorbatiuk, O.; Vasylchenko, О.

    2014-01-01

    Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding speci...

  5. Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA binding protein.

    Science.gov (United States)

    Dai, Haixia; Choe, Woo-Seok; Thai, Corrine K; Sarikaya, Mehmet; Traxler, Beth A; Baneyx, François; Schwartz, Daniel T

    2005-11-09

    We show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable (nonequilibrium) conditions, reproducing a key feature of biological hard-tissue growth and assembly. The nonequilibrium synthesis of Cu(2)O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein TraI in a room-temperature precursor electrolyte. The functional TraI derivative (TraIi1753::CN225) is engineered to possess a cysteine-constrained 12-residue Cu(2)O binding sequence, designated CN225, that is inserted into a permissive site in TraI. When TraIi1753::CN225 is included in the precursor electrolyte, stable Cu(2)O nanoparticles form, even though the concentrations of [Cu(+)] and [OH(-)] are at 5% of the solubility product (K(sp,Cu2O)). Negative control experiments verify that Cu(2)O formation is controlled by inclusion of the CN225 binding sequence. Transmission electron microscopy and electron diffraction reveal a core-shell structure for the nonequilibrium nanoparticles: a 2 nm Cu(2)O core is surrounded by an adsorbed protein shell. Quantitative protein adsorption studies show that the unexpected stability of Cu(2)O is imparted by the nanomolar surface binding affinity of TraIi1753::CN225 for Cu(2)O (K(d) = 1.2 x 10(-)(8) M), which provides favorable interfacial energetics (-45 kJ/mol) for the core-shell configuration. The protein shell retains the DNA-binding traits of TraI, as evidenced by the spontaneous organization of nanoparticles onto circular double-stranded DNA.

  6. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora

    2012-01-01

    Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1ß during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular...... calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca(2+) pathway. The resulting increase in the intracellular calcium concentration...

  7. Protein (Cyanobacteria): 428204399 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available putative calcium-binding protein Pleurocapsa sp. PCC 7327 MALIKGDSFDNNLLGTPNNDTILGFEGKDTLLGLGGNDSLDGGQDSDLLE...GGQGDDTLYSGTLDDDYSSFDLDTLRGGAGNDLLYGEELGYASLLLDGGEGNDTLIGGRGNDTLMGGAGDDYLSAYPSYDEGLWGDDIFYGGSGNDTLIGAWNNTLYG...GNGNDSLDGWFSVGYGGDGDDILSTESNGNDSLYGEGGNDSLNSGASNDTLKGGSGDDTLNGGQYYVYARYRLYTGGGSDF...LDGGAGNDVLYGGFSFEEGNFVYGWDDVDNDTLIGGAGNDTLYGEEDSDSLDGGRGNDLLYGGGERNFLIGDPPYFSLLDLDDTLIGGAGDDTLYGDNGSDRLDGGRG...NDWLYGGGKQNIVDTPNGRVELDANDTLIGGAGNDTLIGMLGNDSLSGGDGADRLDGYGSDPDPDAIDTLSGGRGVDTFVLGNASGAYYLGDGRAIIEDWKANDLLEVYGSLDDYSLDTTANLVGSSAVDTQIFYNSDLIAILQDITNISISDDFVVV

  8. Protein (Cyanobacteria): 493575929 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2232:23 ... putative calcium-binding protein Gloeocapsa sp. PCC 73106 MAIVFGTNNSDSITPIFISAGVSGFPTNGGDLIDALAGNDT...VDGGGGNDSIQGGDGNDSLLGNLGIDTIDGGTGNDSIDGNAGNDFLFGSDGNDTLIGSEGLDFLDGGAGNDSLLGGTGIDVLNGGINNDVLDGGDGNDNLNGDEANDSLNGASGNDLLNGGVGNDT...LLGGTGNDTLNGADGNDTINAGDGTDLVDGSTGNDIVAGNAGNDTVFGNIGNDVVNGGDGV...DFLFGEDGNDSVDGGTGNDFLDGGSGNDSLNGSEGNDTVNGFDGNDLVLGGTGNDSLAGGAGNDTVNGFDGHDFVEGNDNNDSLLGGSGNDTITGGTGNDSLIGGTGN

  9. Protein (Cyanobacteria): 493554048 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available tive calcium-binding protein Xenococcus sp. PCC 7305 MNIQGTTGNDILEGTLEADIIDALAGDDSVIGLAGDDDIVGGLGKDTIRGFGGNDT...IDGGNNNDNLFGDEGDDSITGGDGFDNLGGFTGNDFLNGGLGNDTLSGGAGNDTLIGAEGNDIIFGGADQDILDGGSGSDILKAGSGDDQILGMNGNDVLEGESGADDIFGGEGNDTARGGQDNDT...IDGGNGEDKLFGDFGNDSLLGNQDNDTLVGAEGDDTLVGGTGADQLFGNADRDSLIGGEGND...SMFGGQAADTIIGGNGDDSVFGNLGDDIVKGDVGNDILQGNEGNDSLEGGAGLDTIFGGSENDTLVGNGGADYLDGGSGNDVILAGIDNDIIAGRRGNDQISAGLGNDSALGGNGNDTILGGDGNDT...LNGDANNDVLRGENGDDLLAGGKGSDSLFGDAGNDTLMGAANYVVDPQITELFLDTLIGGSGADTYDFATGLDNFLVDSATLDLGIETAEIRGFAPGQDIFQLSADPNISYGVTQNVTVAEITATIDGNDYLIAQVNGIGNFDLNAAYVQEV

  10. Protein (Cyanobacteria): 218438228 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ... Hemolysin-type calcium-binding protein Cyanothece sp. PCC 7424 MADFILDAQGGILTGTNEADLISFAVNITGQINSLEGNDTI...QSLGGNSVTGTQIILANQGDDLISSASTGGFLTTIEAYGGQGNDTVVGGNFNDTLYGSEGFDTIQGGGGDDSIGGGLQTASQTSDLADTIDGGEGNDIIFGNLGIDTLVGNIGNDT...VFGGRDGDSIDGGLDNDSLVGGEGADTITGGTGNDTLVGGLQTSSQTGDLADNLNGGLGNDVIFGNLGNDTLIGDIGDDILFGGRDNDT...LSGGANNDSLVGGEGADTITGDEGDDTIRGGLQTASQSDDLADSLNGGLGNDLIFGNLGNDTLLGDIGDDILFGGIDNDSIDGGADQDSLVGGENIDTITGGAGNDT...IIGGLQSGDQSNDEADNINGGVGNDSILGNIGVDTLLGDAGNDIIFGGRDNDSIDGGTENDSLIGGEGTDTLVGGNGNDTIIGGLQTADQSGD

  11. A bioinformatic survey of RNA-binding proteins in Plasmodium.

    Science.gov (United States)

    Reddy, B P Niranjan; Shrestha, Sony; Hart, Kevin J; Liang, Xiaoying; Kemirembe, Karen; Cui, Liwang; Lindner, Scott E

    2015-11-02

    The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A repr