Relevant bibliographies by topics / Nucleotide Binding Proteins

Academic literature on the topic 'Nucleotide Binding Proteins'

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Published: 6 September 2023

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Journal articles on the topic "Nucleotide Binding Proteins"

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Katada, Toshiaki, and Michio Ui. "Guanine nucleotide-binding proteins." Japanese Journal of Pharmacology 49 (1989): 26. http://dx.doi.org/10.1016/s0021-5198(19)55959-5.

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Möller, W., and R. Amons. "Phosphate-binding sequences in nucleotide-binding proteins." FEBS Letters 186, no. 1 (July 1, 1985): 1–7. http://dx.doi.org/10.1016/0014-5793(85)81326-0.

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Ye, Zhengmao, John D. Lich, Christopher Moore, Kristi L. Williams, Joseph A. Duncan, and Jenny P. _Y Ting. "ATP-binding to the CATERPILLER protein Monarch-1, is required for its inhibitory function (44.19)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S51. http://dx.doi.org/10.4049/jimmunol.178.supp.44.19.

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Abstract The newly discovered CATERPILLER (NOD, NLR) family is defined by a nucleotide-binding domain (NBD) that consists of the Walker A and B motifs and ten additional motifs. It is conserved from plants to humans, and is important in immune regulation and several genetic disorders. Although the NBD is a defining feature of this family, there is a paucity of data to indicate that these proteins bind nucleotides, or the binding of nucleotides modulate their functions. Monarch-1/Pypaf7 is a negative regulator of IRAK-1 and NIK. In this report, we successfully purified recombinant Monarch-1 proteins to homogeneity. Purified Monarch-1 binds ATP but not CTP, GTP and UTP and exhibits ATP hydrolysis activity. Intact Walker A/B sequences are required for nucleotide binding. Overexpression of Monarch-1 nucleotide-binding defective mutant in THP-1 cells resulted in a dramatic increase of IL-6, chemokine CCL1, CXCL6 and CXCL13 to the extent that is comparable to the THP-1 Monarch-1 shRNA knock down cells in response to TLR2 agonist. This indicates that ATP binding is required for the inhibitory function of Monarch-1 and the nucleotide binding-defective mutant displays a dominant negative effect. Intact nucleotide-binding domain is also required for IRAK-1 dephosphorylation and p100 processing suggesting a nucleotide binding-dependent mechanism of Monarch-1 function in both canonical and non-canonical NF-κB pathway.
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Hatley, M. E., S. W. Lockless, S. K. Gibson, A. G. Gilman, and R. Ranganathan. "Allosteric determinants in guanine nucleotide-binding proteins." Proceedings of the National Academy of Sciences 100, no. 24 (November 17, 2003): 14445–50. http://dx.doi.org/10.1073/pnas.1835919100.

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Milligan, G. "Guanine nucleotide binding proteins and cellular control." Current Opinion in Cell Biology 1, no. 2 (April 1989): 196–200. http://dx.doi.org/10.1016/0955-0674(89)90086-0.

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Spiegel, Allen M. "Signal transduction by guanine nucleotide binding proteins." Molecular and Cellular Endocrinology 49, no. 1 (January 1987): 1–16. http://dx.doi.org/10.1016/0303-7207(87)90058-x.

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Davis, Beckley, Suzanna Talento, Aaron Tocker, and Joseph Duncan. "Nucleotide binding characteristics of NOD2 proteins (INM1P.442)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 56.19. http://dx.doi.org/10.4049/jimmunol.194.supp.56.19.

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Abstract Nucleotide binding and oligomerization domain containing protein 2 (NOD2) is an intracellular protein that is involved in the recognition of bacterial cell wall derived muramyl-dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders including Crohn’s disease and Blau syndrome. NOD2 is a member of the Nucelotide-binding domain and leucine-rich repeat containing protein gene family (NLR). Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by pathogenic NOD2 proteins remains unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl-dipeptide in cellular assay. We demonstrate that nucleotide binding is necessary for wild type and pathogenic NOD2 protein activation of the NF-κB signaling pathway. Mutations of the Walker A motif inhibit binding to RIP2k in wild type NOD2, Crohn’s disease-associated NOD2 and Blau syndrome-associated NOD2. Loss of RIP2k binding correlates with decreased NF-κB-dependent luciferase activity. These data suggest that nucleotide binding is necessary for NOD2 function.
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Cheng, Y. S., C. E. Patterson, and P. Staeheli. "Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP." Molecular and Cellular Biology 11, no. 9 (September 1991): 4717–25. http://dx.doi.org/10.1128/mcb.11.9.4717-4725.1991.

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The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN-induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN-induced protein previously shown to be absent from mice of Gbp-1b genotype.
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Cheng, Y. S., C. E. Patterson, and P. Staeheli. "Interferon-induced guanylate-binding proteins lack an N(T)KXD consensus motif and bind GMP in addition to GDP and GTP." Molecular and Cellular Biology 11, no. 9 (September 1991): 4717–25. http://dx.doi.org/10.1128/mcb.11.9.4717.

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The primary structures of interferon (IFN)-induced guanylate-binding proteins (GBPs) were deduced from cloned human and murine cDNAs. These proteins contained only two of the three sequence motifs typically found in GTP/GDP-binding proteins. The N(T)KXD motif, which is believed to confer guanine specificity in other nucleotide-binding proteins, was absent. Nevertheless, the IFN-induced GBPs exhibited a high degree of selectivity for binding to agarose-immobilized guanine nucleotides. An interesting feature of IFN-induced GBPs is that they strongly bound to GMP agarose in addition to GDP and GTP agaroses but failed to bind to ATP agarose and all other nucleotide agaroses tested. Both GTP and GMP, but not ATP, competed for binding of murine GBP-1 to agarose-immobilized GMP. The IFN-induced GBPs thus define a distinct novel family of proteins with GTP-binding activity. We further demonstrate that human and murine cells contain at least two genes encoding IFN-induced GBPs. The cloned murine cDNA codes for GBP-1, an IFN-induced protein previously shown to be absent from mice of Gbp-1b genotype.
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Manji, Husseini K., Guang Chen, William Potter, and Thomas R. Kosten. "Guanine nucleotide binding proteins in opioid-dependent patients." Biological Psychiatry 41, no. 2 (January 1997): 130–34. http://dx.doi.org/10.1016/s0006-3223(96)00216-8.

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Dissertations / Theses on the topic "Nucleotide Binding Proteins"

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Tisi, Dominic John Guiseppe. "Structural studies on nucleotide binding proteins." Thesis, Birkbeck (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391822.

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Johansson, Kenth. "Structural studies of four nucleotide binding proteins : aldehyde dehydrogenase, NADP-malate dehydrogenase and two deoxynucleoside kinases /." Uppsala : Swedish University of Agricultural Sciences, 2000. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=009416200&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.

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Mutomba, Martha Chengetai. "Guanine nucleotide-binding proteins of Trypanosoma brucei." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308280.

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Iuga, Adriana. "Solid-state 31P NMR of nucleotide binding proteins." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973225238.

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Heurtel, Thuswaldner Sophie. "Nucleotide-binding Proteins in the Plant Thylakoid Membrane." Licentiate thesis, Linköping Department of Biomedicine and Surgery, Linköping University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-7934.

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Worth, Graham Alan. "The energetics of nucleotide binding to RAS proteins." Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:44524415-2f2b-4601-998c-56110f332153.

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Ras proteins are a special class of proteins that mediate cell growth signals. Their importance lies in the fact that they are products of a proto-oncogene. This means that under certain conditions the gene that determines its structure is altered and a mutant protein results that is involved in the transformation of normal cells to cancer cells. The actual function by which the protein acts in the signal pathway is not known. However it is known that they act as a switch, undergoing a cycle involving the exchange of guaninosine nucleotides in the binding site. This thesis uses computer simulations to study the energetics of this binding, with the long term aim of developing a drug to inhibit the transforming activity of the oncogenic protein. To begin with, a model of the protein based on a crystal structure is built. Using Molecular dynamics the motion of this model is studied. A possible mechanism by which one half of the nucleotide cycle could be induced is investigated, with the result that phosphorylation of the protein may be involved. The main part of the thesis is then devoted to using the free energy perturbation (FEP) method to calculate the difference in Gibbs binding free energy between the nucleotides in the protein. Using histamine as a model, a method of dealing with charged, flexible molecules is developed; namely the inclusion of a reaction field and comprehensive conformational analysis. The results from the associated calculations are seen to be very close to experimental data. The same procedures are then applied to the much more complex ras: nucleotide system with less successful results, the reason for which is mostly due to the restriction of limited computer resources to tackle such a problem. The conclusion is that given the resources and by using the techniques developed in this thesis, this type of calculation is a feasible way to study such systems.
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Bramble, Sharyl Elizabeth. "Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium discoideum." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26172.

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One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucleotides like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography. A number of different approaches were used to determine guanine nucleotide binding by p23RAS . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. discoideum cells. In contrast p21RAS from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the presence of ³²P-α-guanosine 5¹-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23RAS has a very low affinity for guanine nucleotides such that GTP binding was not detectable in these experiments or that the ras protein from D. discoideum simply does not bind guanine nucleotides. The purification of p23RAS from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti-ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be enriched relative to other proteins because the immunoaffinity columns did not bind p23RAS.
Science, Faculty of
Microbiology and Immunology, Department of
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Law, Wing-lun, and 羅永倫. "Expression, purification and preliminary x-ray crystallographic studies of two nucleotide binding proteins." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46939118.

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Akam, Elizabeth Claire. "The activation of guanine nucleotide binding proteins by muscarinic acetylcholine receptor subtypes." Thesis, University of Leicester, 1999. http://hdl.handle.net/2381/29919.

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Agonist-stimulation of human recombinant M1, M2, M3 and M4 receptors, expressed in Chinese hamster ovary cells, was investigated at the level of G protein activation. Functional responses were determined by a number of methods including [35S]-GTPS binding in membranes using both filtration-based and immunoprecipitation-based procedures: Ins(1,4,5)P3 accumulation and 45Ca2+ release from permeabilised cell suspensions; and cAMP accumulation in cell suspensions. M2 and M4 receptors, with equivalent expression levels in this recombinant system, were found only to couple to pertussis toxin-sensitive G proteins with near equal kinetics. Methacholine appeared equipotent when activating the total G protein complement through the M2 and M4 receptors, however, it appeared more potent when activating Gi3/o through the M2 compared to the M4 muscarinic receptor. Using equivalent expression levels of M1 and M3 receptors both the subtypes were found to couple to both pertussis toxin-sensitive and -insensitive G proteins. CHO-M1 and -M3 mediated Ins(1,4,5)P3 generation after pertussis toxin pre-treatment suggested the functional significance of coupling to multiple G protein classes may be in the stimulation of PLC by -subunits derived from Gi-like G proteins. The activation of Gq/11 through the M1 receptor subtype, after methacholine-stimulation, is faster, greater and more potent than that mediated by the M3 receptor subtype, suggesting that the intrinsic activity of the M1 subtype is greater than that of the M3 subtype. The 'partial' agonist pilocarpine also displayed very different G protein activation profiles after stimulation of M1, M2, M3 and M4 receptor subtypes, suggesting that agonists acting at different receptor subtypes may be capable of inducing relatively selective coupling of the occupied receptor to available G proteins. This study therefore concludes that muscarinic receptor subtypes display divergent G protein activation profiles after either 'full' or 'partial' agonist-stimulation.
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Wadman, Isobel A. "The regulation of human platelet adenylate cyclase by ATP and guanine nucleotide binding proteins." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241124.

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Books on the topic "Nucleotide Binding Proteins"

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Bosch, L., B. Kraal, and A. Parmeggiani, eds. The Guanine — Nucleotide Binding Proteins. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2.

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EMBO-NATO-CEC Advanced Research Workshop on the Guanine-Nucleotide Binding Proteins: Common Structural and Functional Properties (1988 Renesse, Netherlands). The guanine-nucleotide binding proteins: Common structural and functional properties. New York: Plenum Press, 1989.

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Taylor, Catherine Yvonne. Analysis of protein binding motifs in the nucleotide sequence of the human [gamma]-actin gene promoter. Ottawa: National Library of Canada = Bibliothèque nationale du Canada, 1993.

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NATO, Advanced Research Institute on Biological Signal Transduction (1990 Island of Spetsai Greece). Biological signal transduction. Berlin: Springer-Verlag, 1991.

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The Guanine - Nucleotide Binding Proteins:Common Structural and Functional Properties. Springer, 1989.

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Bosch, L. Guanine -- Nucleotide Binding Proteins: Common Structural and Functional Properties. Springer, 2013.

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Bosch, L. The Guanine - Nucleotide Binding Proteins: Common Structural And Functional Properties. Springer, 2013.

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Han, Hyung Mee. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha₁-adrenergic system. 1989.

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Book chapters on the topic "Nucleotide Binding Proteins"

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Mandelkow, Eva-Maria, Klaus Linse, and Eckhard Mandelkow. "Tubulin Structure and Nucleotide Binding." In The Guanine — Nucleotide Binding Proteins, 385–90. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_38.

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Dever, Thomas E., and William C. Merrick. "The GTP-Binding Domain Revisited." In The Guanine — Nucleotide Binding Proteins, 35–48. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_4.

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Nyborg, J., and T. la Cour. "New Structural Data on Elongation Factor-Tu:Gdp Based on X-Ray Crystallography." In The Guanine — Nucleotide Binding Proteins, 3–14. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_1.

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Peter, Marcus E., and Mathias Sprinzl. "Affinity Labeling of the GDP/GTP Binding Site in Thermus Thermophilus Elongation Factor Tu." In The Guanine — Nucleotide Binding Proteins, 99–110. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_10.

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Jonák, Jiří, Karel Karas, and Ivan Rychlík. "Characterization of Elongation Factor Tu from Bacillus Subtilis Modified by Affinity Labelling." In The Guanine — Nucleotide Binding Proteins, 111–19. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_11.

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Kraal, Barend, Jan Pieter Abrahams, and Leendert Bosch. "Effects of Kirromycin on the Elongation Factor EF-Tu and its Interactions with GDP or GTP and tRNA. The Application of zone-Interference Gel Electrophoresis, a New Method for the Analysis of Weak Complexes." In The Guanine — Nucleotide Binding Proteins, 121–29. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_12.

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Möller, Wim, and Reinout Amons. "Factors and Ribosomes: Their Coupling and Mode of Signal Processing." In The Guanine — Nucleotide Binding Proteins, 131–42. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_13.

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Hershey, John W. B., Vinay K. Pathak, Heidemarie Ernst, Markus Hümbelin, and Randal J. Kaufman. "The Structure and Regulation of Mammalian Initiation Factor eIF2." In The Guanine — Nucleotide Binding Proteins, 143–50. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_14.

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Chardin, P., N. Touchot, A. Zahraoui, V. Pizon, I. Lerosey, B. Olofsson, and A. Tavitian. "Structure of the Human ras Gene Family." In The Guanine — Nucleotide Binding Proteins, 153–63. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_15.

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Willumsen, Berthe M., Hedy Adari, Ke Zhang, Alex G. Papageorge, James C. Stone, Frank McCormick, and Douglas R. Lowy. "A Mutational Analysis of ras Function." In The Guanine — Nucleotide Binding Proteins, 165–77. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4757-2037-2_16.

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Conference papers on the topic "Nucleotide Binding Proteins"

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Liao, Jung-Chi, and George Oster. "The Engines of Biomolecular Motors." In ASME 2004 3rd Integrated Nanosystems Conference. ASMEDC, 2004. http://dx.doi.org/10.1115/nano2004-46094.

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The majority of biomolecular motors are powered by nucleoside triphosphate (NTP), especially adenosine triphosphate (ATP). These motors consist of a β-sheet with highly conserved motifs and the nucleotide binding domain around it. The highly conserved protein folds are the engines of these motors, which convert the energy of NTP hydrolysis cycle to mechanical work. Although functions of molecular motors are widely diverse, (including cargo movement, DNA unwinding, protein degradation, ion pumping, etc), the nucleotide binding domains are very similar. In the binding site, NTP undergoes a hydrolysis cycle E+NTP⇄E·NTP⇄E•NTP⇄E•NDP•Pi⇄E•NDP+Pi⇄E+NDP+Pi where E is the enzyme (motor protein), the small dot represents the docking of NTP, and the large dot represents the tightly-bound states. The hydrogen bond network formed in the NTP binding step, as shown in Figure 1 [1], deforms the β-sheet and adjacent structures. The local deformation propagates to conformational changes of functional residues to do mechanical work or to change the affinity to the substrate [2]. For multimeric motor proteins, we must also consider the stress paths among subunits which control the sequence and the activity of the protein. Stress trajectories emanating from a binding site either passes through a circumferential stress loop or a stress loop through the substrate.
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Lapetina, Eduardo G. "THE ROLE OF INOSITIDES, PHOSPHOLIPASE C AND G-PROTEINS IN RECEPTOR TRANSDUCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644775.

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It is now widely recognized that the activation of phospholipase C by specific agonists leads to the formation of two second messengers: (1) inositol trisphosphate, which releases Ca2+ from the endoplasmic reticulum to the cytosol and (2) 1,2- diacylglycerol, which stimulates protein kinase C. In the past few years, GTP-binding proteins have been associated with the regulation of phospholipase C. However, the identity of the GTP-binding protein involved and the type of association with phospholipase C is not yet known. It is now recognized that there are two types of phospholipase C enzymes: (a) a soluble enzyme that has been characterized in several tissues and does not preferentially hydrolyze polyphospholinositides and (b) membrane-bound enzymes that are coupled to the receptors, specifically hydrolyzing polyphosphoinositides and activated by membrane guanine nucleotide-binding proteins. Recent reports have tried to assess the involvement of GTP-binding proteins in the agonist-induced stimulation of phospholipase C, and various related aspects have been reported. These are concerned with: (a) detection of various GTP-binding proteins in platelets, (b) the effects of known inhibitors of GTP-binding proteins such as GDPgS or pertussis toxin on the agonist-induced stimulation of phospholipase C, (c) the direct effects of stimulators of GTP-binding proteins such as GTP, GTP-analogs and fluoride on phospholipase C activity, (d) the possible association of GTP-binding proteins to cytosolic phospholipase C that would then lead to degradation of the membrane-bound inositides and (e) cytosolic phospholipase C response to the activation of cell surface receptors. The emerging information has had contradictory conclusions. (1) Pretreatment of saponin-permeabilized platelets with pertussis toxin has been shown to enhance and to inhibit the thrombin-induced activation of phospholipase C. Therefore, it is not clear if a G protein that is affected by pertussis toxin in a manner similar to Gi or Go plays a central role in activation of phospholipase C. (2) Studies on the effect of GDPβ;S are also conflicting indicating that there may be GTP-independent and/or -dependent pathways for the activation of phosphoinositide hydrolysis. (3) A cytosolic phospholipase C is activated by GTP, and it has been advanced that this activity might trigger the hydrolysis of membrane-bound inositides. A cytosolic GTP-binding protein might be involved in this action, and it is speculated that an α-subunit might be released to the cytoplasm by a receptor-coupled mechanism to activate phospholipase C. However, no direct evidence exists to support this conclusion. Moreover, the exact contribution of phospholipase C from the membranes or the cytosol to inositide hydrolysis in response to cellular agonists and the relationship of those activites to membrane-bound or soluble GTP-binding proteins are unknown. Our results indicate that the stimulation of phospholipase C in platelets by GDPβS and thrombin are affected differently by GDPβS. GDPgSinhibits the formation of inositol phosphates produced by GTPγS but not that induced by thrombin. Thrombin, therefore, can directly stimulate phospholipase C without the involvement of a “stimulatory” GTP-binding protein, such as Gs, for the agonist stimulation of adenylate cyclase. However, an “inhibitory” GTP-binding protein might have some influence on thrombin-stimulated phospholipase C, since in the presence of GDPγS thrombin produces a more profound stimulation of phospholipase C.This “inhibitory” GTP-binding protein might be ADP-ribosylated by pertussis toxin because pertussis toxin can also enhance thrombin action on phospholipase C activity. Therefore, phospholipase C that responds to thrombin could be different from the one that responds to GTPγS. Cytosolic phospholipase C can be activated by GTP or GTP analogs, and the one that responds to thrombin should be coupled to the receptors present in the plasma membrane. The initial action of thrombin is to directly activate the plasma membrane-bound phospholipase C and the mechanism of this activation is probably related to the proteolytic action of thrombin or the activation of platelet proteases by thrombin. In agreement with this, trypsin can also directly activate platelet phospholipase C and, subsequently, GTPyS produces further activation of phospholipase C. If these two mechanisms are operative in platelets, the inhibition of cytosolic phospholipase C by GDPβS would allow a larger fraction of inositides for degradation of the thrombin-stimulated phospholipase C, as our results show.
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Jakobs, K. H., P. Gierschik, and R. Grandt. "THE ROLE OF GTP-BINDING PROTEINS EXHIBITING GTPase ACTIVITY IN PLATELET ACTIVATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644773.

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Activation of platelets by agonists acting via cell surface-located receptors apparently involves as an early event in transmembrane signalling an interaction of the agonist-occupied receptor with a guanine nucleotide-binding regulatory protein (G-protein). The activated G-protein, then, transduces the information to the effector molecule, being responsible for the changes in intracellular second messengers. At least two changes in intracellular signal molecules are often found to be associated with platelet activation by agonists, i.e., increases in inositol trisphosphate and diacylglycerol levels caused by activation of a polyphosphoinositide-specific phospholipase C and decrease in cyclic AMP concentration caused by inhibition of adenylate cyclase.Both actions of platelet-activating agents apparently involve G-proteins as transducing elements. Generally, the function of a G-protein in signal transduction can be measured either by its ability to regulate the activity of the effector molecule (phospholipase C or adenylate cyclase) or the binding affinity of an agonist to its specific receptor or by the abitlity of the G-protein to bind and hydrolyze GTP or one of its analogs in response to agonist-activated receptors. Some platelet-activating agonists (e.g. thrombin) can cause both adenylate cyclase inhibition and phospholipase C activation, whereas others induce either inhibition of adenylate cyclase (e.g. α2-adrenoceptor agonists) or activation of phospholipase C (e.g. stable endoperoxide analogs) . It is not yet known whether the simultaneous activation of two signal transduction systems is due to activation of two separate G-proteins by one receptor, to two distinct receptors activating each a distinct G-protein or to activation of two effector molecules by one G-protein.For some of the G-proteins, rather specific compounds are available causing inactivation of their function. In comparison to Gs, the stimulatory G-protein of the adenylate cyclase system, the adenylate cyclase inhibitory Gi-protein is rather specifically inactivated by ADP-ribosylation of its a-subunit by pertussis toxin, “unfortunately” not acting in intact platelets, and by SH-group reactive agents such as N-ethylmaleimide and diamide, apparently also affecting the Giα-subunit. Both of these treatments completely block α2-adrenoceptor-induced GTPase stimulation and adenylate cyclase inhibition and also thrombin-induced inhibition of adenylate cyclase. In order to know whether the G-protein coupling receptors to phospholipase C is similar to or different from the Gi-protein, high affinity GTPase stimulation by agents known to activate phospholipase C was evaluated in platelet membranes. The data obtained indicated that GTPase stimulation by agents causing both adenylate cyclase inhibition and phospholipase C activation is reduced, but only partially, by the above mentioned Gi-inactivating agents, while stimulation of GTPase by agents stimulating only phospholipase C is not affected by these treatments. These data suggested that the G-protein regulating phospholipase C activity in platelet membranes is different from the Gi-protein and may also not be a substrate for pertussis toxin. Measuring thrombin stimulation of inositol phosphate and diacylglycerol formation in saponin-permeabilized platelets, apparently contradictory data were reported after pertussis toxin treatment, being without effect or causing even an increase in thrombin stimulation of inositol phosphate formation (Lapetina: BBA 884, 219, 1986) or being inhibitory to thrombin stimulation of diacylglycerol formation (Brass et al.: JBC 261, 16838, 1986). These data indicate that the nature of the phospholipase C-related G-protein(s) is not yet defined and that their elucidation requires more specific tools as well as purification and reconstitution experiments. Preliminary data suggest that some antibiotics may serve as useful tools to characterize the phospho-lipase-related G-proteins. The possible role of G-protein phosphorylation by intracellular signal molecule-activated protein kinases in attenuation of signal transduction in platelets will be discussed.
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Stenflo, J., A.-K. öhlin, Å. Lundvall, and B. Dahlback. "β-HYDROXY ASPARTIC ACID AND ft-HYDROXYASPARAGINE IN THEEGF-HOMOLOGY REGIONS OF PROTEIN C AND PROTEINS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643995.

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The amino acid sequence has been determined for all of the vitamin K-dependent proteins and the gene structure is known for some of them. These findings have shown the proteins to consist of four clearly discernible domains, except protein S which has six domains. The protein domains seem to be coded on separate exons (Foster, D. C. et. al. 1985 Proc. Natl. Acad. Sci. USA 82,4673). The vitamin K-dependent γ-carboxyglutamic acid (Gla) containing domain isthe common structural denominator of the members of this protein family. In addition, all of these proteins except prothrombin contain domains that are homologous to the precursor of the epidermal growth factor (EGF). Such domains arealso found in proteins that are not vitamin K-dependent, such as the low density lipoprotein receptor, thrombomodulin, factor XII, plasminogen, the tissue type plasminogen activator, urokinase and the complement protein Clr. The vitamin K-dependent proteins can be dividedinto three groups. Factors VII, IX, X, protein C and protein Z form one group, which in addition to the Gla-region have two EGF-homology regions and one domain that is homologous to the serine proteases. Prothrombin has two 'kringle' structures and a serine protease domain and constitutes a group of its own. Protein S is also unique in that it has four EGF-homology regions and a COOH-terminal region that is homologous to the sexual hormone binding globulin (see poster by Edenbrand et. al.).Recently a posttranslationally modified amino acid, B-hydroxyaspatic acid (Hya), was identified in position 71 in the NH2-terminal EGF-homology region ofbovine protein C. The amino acid is formed by hydroxylation of aspartic acid. It has also been identified in the corresponding positions in factors VII, IX,X and protein Z (i. e. proteins which like protein C have two EGF-homology regions each). In protein S the N2-terminal of four EGF-homology regions has hydroxy lated aspartic acid .whereas the following three EGF-like domains have B-hydroxyasparagine. The nucleotide sequence codes for asparagine in the three latter positions. Neither vitamin K nor vitamin C seem to be involvedin the formation of the two hydroxylated amino acids. Recently, Hya was identified in acid hydrolysates of the complement protein Clr. Hya and Hyn have onlybeen found in domains that are homologous to the EGF precursor. In an attempt to identify the structural requirement of the hydroxylating enzyme, we have compared the sequences of EGF-homology regions that contain Hya or Hyn with the corresponding sequences that have been shown not to contain the modified amino acids. The domains that have Hya or Hyn have the consensus sequence Cx xxxx xCxC. This sequence has been found in three EGF-like domains in the EGF-precursor, in two in the LDL-receptor and in two in thrombomodulin. Furthermore, the neurogenic Notch locus in Drosophila melanogaster codes for 36 EGF-homolgy regions, 22 of which contain the consensussequence, whereas the Lin-12 locus in Caenorhabditis elegans codes for at least 11 EGF-like repeats, two of which comply with the consensus sequence. Whether any of these proteins contain Hya orHyn is not yet known with certainty.It has been hypothesized that Hya isinvolved in the Gla independent Ca2+binding of factors IX, X and protein C. In an attempt to resolve this issue, we have isolated the EGF-homology region from human protein C and been able to demonstrate that it binds Ca2+ (see poster by öhlin and Stenflo). However, we do not yet know whether Hya is directly involved in the Ca2+binding.
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Brass, L. F., D. R. Manning, and M. J. Woolkalis. "G PROTEIN REGULATORS OF PHOSPHOLIPASE C AND ADENYLATE CYCLASE IN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644630.

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The hydrolysis of polyphosphoinositides (PI) by phospholipase C during platelet activation produces two key intracellular messengers, inositol triphosphate and diacylglycerol. This process is thought to be regulated by a guanine nucleotide binding protein referred to as Gp. Although the evidence that Gp exists is compelling, to date it has not been isolated. Uncertainty about its identity has been compounded by variations between tissues in the susceptibility of Gp to pertussis toxin and by reconstitution studies which show that pertussis toxin-inhibited PI hydrolysis can be restored by purified Gi, the pertussis toxin-sensitive G protein which inhibits adenylate cyclase. Therefore, it remains unclear whether Gp represents a new G protein or a second role for Gj. When platelets permeabilized with saponin were incubated with pertussis toxin and 32P-NAD, a single 42 kDa protein was 32P-ADP-ribosylated which co-migrated with the purified a subunit of Gi. Preincubating the platelets with an agonist inhibited labeling of this protein by dissociating the G protein into subunits. The extent of inhibition correlated with the number of toxin-sensitive functions caused by the agonist. Labeling was abolished by thrombin, which inhibited cAMP formation and caused toxin-inhibitable PI hydrolysis. Labeling was partially inhibited by vasopressin and platelet activating factor, which caused toxin-inhibitable PI hydrolysis, but had no effect on cAMP formation and by epinephrine, which inhibited cAMP formation, but did not cause PI hydrolysis. Labeling was unaffected by the TxA2 analog U46619, which neither caused toxin-sensitive PI hydrolysis nor inhibited cAMP formation. These observations suggest that the 42 kDa band may contain a subunits from both Gp and Gi and, in fact, 2D electrophoresis resolved the 42 kDa protein band into two proteins with distinct pi. However, those agonists linked functionally only to Gp or only to Gi decreased the labeling of both proteins. Therefore, our data suggest (1) that Gj and Gp are the same protein and (2) that whether a aiven platelet agonist affects adenylate cyclase or phospholipase C or both depends upon factors extrinsic to the G protein.
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Kienast, J., J. Arnout, G. Pfliegler, H. Deckmyn, E. Van Houtte, and J. Vermylen. "DUAL EFFECT OF FLUORIDE ON ENDOTHELIAL PROSTACYCLIN PRODUCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643377.

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The effects of fluoride at mM concentrations on prostacyclin (PGI2) production by human umbilical vein endothelial cells in culture were investigated. At lower concentrations, with a peak activity around 20-30 mM, sodium fluoride (NaF) caused a slow rise in PGI2 release reaching a maximum of 6 to 8 × control values after 1 hour. With increasing concentrations, the NaF-induced PGI2 production progressively decreased to almost control levels at 100 mM. Preincubation of the cells with 100 mM NaF inhibited thrombin— and A23187-stimulated PGI2 production by 46 % and 64 % respectively. The response to NaF was not significantly enhanced by AlCl3 nor was it inhibited by pertussis toxin.The dual, stimulatory and inhibitory, effect of NaF on endothelial PGI2 production as well as its slow mode of action resemble the stimulus-response pattern observed in platelets upon exposure to NaF as it has previously been described by our grcup. Therefore, NaF might be an interesting tool for the investigation of intracellular regulatory processes triggering endothelial PGI2 production, particularly in view of the known capacity of fluoride to activate guanine nucleotide—binding regulatory proteins.
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Authi, K. S., B. J. Evenden, and N. Crawford. "ACTION OF GTPγS [GUANOSINE 5∲-0-(3-THIOPHOSPHATE)] ON SAPONIN-PERMEABILISED PLATELETS: INVOLVEMENT OF 'G' PROTEINS IN PLATELET ACTIVATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644514.

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Certain ligand-receptor interactions at cell surfaces lead to the phospholipase-C (PLC) hydrolysis of phosphatidyl inositol (4.5) bisphosphate (PIP2). The products serve as intracellular second messengers, e.g. inositol (1.4.5) trisphosphate (IP3) releases Ca2+ from intracellular stores and diacylglycerol activates protein kinase-C. From studies using GTP and analogues (e.g. GTPγS) there is evidence of a key role for a guanine nucleotide binding protein(s) as a link between receptors and PIP2 hydrolysis. We report the actions of GTPγS on washed human platelets permeabilised with saponin (12-14 μg/ml) to allow penetration of low MWt polar substances. The responses to GTPγS are dose dependent (range 9-60 μM) and at 60 μM the agent induces shape change, aggregation and the secretion of 50% of previously incorporated [14C]-5HT. No effect of GTPγS is seen with intact cells. Shape change occurs 25-30 sec after GTPγS; aggregation and secretion is complete after 3 min. When GTP was used (up to 135 μM) with similarly permeabilised platelets no responses were initiated. Phosphatidylinositol turnover was monitored using 32P-labelling before permeabilisation. The addition of 90 μM GTPγS resulted in a 143 ± 23% (n=4) increase in 32P-phosphatidic acid (PA) with respect to the basal levels of “saponised control” cells. These findings suggest that GTPγS stimulates PLC activity through a ‘G’ protein interaction. The GDP analogue (GDPβS) produced no activation responses in saponised platelets but inhibited responses induced by GTPγS in a dose dependent manner (0-480 μM, max inhibition 480 μM). At 960 μM, GDPβS totally inhibited aggregation and secretion initiated by low doses of thrombin (0.1 U/ml) and collagen (1 μg/ml). Identical inhibition by GDPβS of thrombin and collagen-induced activation of intact platelets was observed indicating membrane penetration of this analogue. Shape change effects were not inhibited by GDPSS. The inhibitory effects of GDPSS towards thrombin and collagen induced secretion could be progressively overcome at higher doses of thrombin (0.2 U/ml - 2 U/ml) and collagen (5 μg/ml - 60 μg/ml) suggesting that at higher concentrations these agonists may exert effects through 'G' protein-independent mechanisms.
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8

Ueno, H., T. Yasunaga, C. Shingyoji, T. Yamaguchi, and K. Hirose. "Dynein Pulls Microtubules Without Rotating Its Stalk." In ASME 2009 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2009. http://dx.doi.org/10.1115/sbc2009-206430.

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Dynein is a motor protein that hydrolyses ATP and moves toward the minus end of a microtubule (MT). A dynein molecule has one to three heavy chains, each consisting of three domains: a head, a stalk and a tail. ATP is bound and hydrolysed in the head, which has a ring-like structure composed of 6 AAA+ domains. The stalk is an antiparallel coiled-coil, 10–15 nm long, and has a nucleotide-dependent MT-binding domain at the tip (1) (Fig. 1). It has been proposed that the nucleotide-dependent binding affinity of the tubulin-binding site at the tip of the stalk is modulated by the two alpha helices in the coiled-coil sliding over each other (2). However, it is not known how a dynein molecule moves along a microtubule (MT).
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Hofmann, Klaus P. "Visual process in retinal photoreceptors: analysis by intrinsic light scattering signals." In OSA Annual Meeting. Washington, D.C.: Optica Publishing Group, 1989. http://dx.doi.org/10.1364/oam.1989.wd1.

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The rod photoreceptor of the retina is a quantum detector whose physiological function in physically unfavorable conditions (body temperature, salt solution) is made possible by specific protein interactions. Photon energy is stored by the receptor protein rhodopsin (R) in a structurally transformed state. Activated R interacts with transducin (a G-protein or guanine nucleotide binding protein). This catalyses binding to G of energy-rich nucleotide which in turn releases G in an activated form. Absorption of one photon leads to the activation of 1000 G in 1 s. Analogous relay systems are found from bacteria to man. Intrinsic physical properties of the rhodopsin G-protein system allow photometric studies in situ and in real time. Activation of R and interaction with G are measurable by absorption spectrophotometry. Activation of G is measurable by light scattering (LS) changes (signals) arising from the shift of the G-protein mass during activation. A continuous transretinal, near infrared LS probing beam affords direct monitoring of G-activation induced by visual stimuli. These optical techniques, combined with biochemical and physiological approaches, have been used to study the sites of R-G interaction and the thermodynamics of the G-relay in situ. G-activation is not modulated by previous illumination, indicating a remarkable constancy of the R-G amplification step in the visual transduction pathway.
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Holden, Todd, G. Tremberger, Jr., E. Cheung, R. Subramaniam, N. Gadura, P. Schneider, R. Sullivan, A. Flamholz, D. Lieberman, and T. D. Cheung. "Nucleotide fluctuation of radiation-resistant Halobacterium sp. NRC-1 single-stranded DNA-binding protein (RPA) genes." In SPIE Optical Engineering + Applications, edited by Richard B. Hoover, Gilbert V. Levin, Alexei Y. Rozanov, and Kurt D. Retherford. SPIE, 2009. http://dx.doi.org/10.1117/12.825827.

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Reports on the topic "Nucleotide Binding Proteins"

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Avni, Adi, and Gitta L. Coaker. Proteomic investigation of a tomato receptor like protein recognizing fungal pathogens. United States Department of Agriculture, January 2015. http://dx.doi.org/10.32747/2015.7600030.bard.

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Maximizing food production with minimal negative effects on the environment remains a long-term challenge for sustainable food production. Microbial pathogens cause devastating diseases, minimizing crop losses by controlling plant diseases can contribute significantly to this goal. All plants possess an innate immune system that is activated after recognition of microbial-derived molecules. The fungal protein Eix induces defense responses in tomato and tobacco. Plants recognize Eix through a leucine-rich-repeat receptor- like-protein (LRR-RLP) termed LeEix. Despite the knowledge obtained from studies on tomato, relatively little is known about signaling initiated by RLP-type immune receptors. The focus of this grant proposal is to generate a foundational understanding of how the tomato xylanase receptor LeEix2 signals to confer defense responses. LeEix2 recognition results in pattern triggered immunity (PTI). The grant has two main aims: (1) Isolate the LeEix2 protein complex in an active and resting state; (2) Examine the biological function of the identified proteins in relation to LeEix2 signaling upon perception of the xylanase elicitor Eix. We used two separate approaches to isolate receptor interacting proteins. Transgenic tomato plants expressing LeEix2 fused to the GFP tag were used to identify complex components at a resting and activated state. LeEix2 complexes were purified by mass spectrometry and associated proteins identified by mass spectrometry. We identified novel proteins that interact with LeEix receptor by proteomics analysis. We identified two dynamin related proteins (DRPs), a coiled coil – nucleotide binding site leucine rich repeat (SlNRC4a) protein. In the second approach we used the split ubiquitin yeast two hybrid (Y2H) screen system to identified receptor-like protein kinase At5g24010-like (SlRLK-like) (Solyc01g094920.2.1) as an interactor of LeEIX2. We examined the role of SlNRC4a in plant immunity. Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defense responses while silencing SlNRC4 reduces plant immunity. We propose that SlNRC4a acts as a non-canonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perception. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 inVHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune . The interaction of LeEIX2 with SlRLK-like was verified using co- immunoprecipitation and a bimolecular fluorescence complementation assay. The defence responses induced by EIX were markedly reduced when SlRLK-like was over-expressed, and mutation of slrlk-likeusing CRISPR/Cas9 increased EIX- induced ethylene production and SlACSgene expression in tomato. Co-expression of SlRLK-like with different RLPs and RLKs led to their degradation, apparently through an endoplasmic reticulum-associated degradation process. We provided new knowledge and expertise relevant to expression of specific be exploited to enhance immunity in crops enabling the development of novel environmentally friendly disease control strategies.
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Fluhr, Robert, and Maor Bar-Peled. Novel Lectin Controls Wound-responses in Arabidopsis. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7697123.bard.

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Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.
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Joel, Daniel M., Steven J. Knapp, and Yaakov Tadmor. Genomic Approaches for Understanding Virulence and Resistance in the Sunflower-Orobanche Host-Parasite Interaction. United States Department of Agriculture, August 2011. http://dx.doi.org/10.32747/2011.7592655.bard.

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Oroginal Objectives: (i) identify DNA markers linked to the avirulence (Avr) locus and locate the Avr locus through genetic mapping with an inter-race Orobanche cumana population; (ii) develop high-throughput fingerprint DNA markers for genotypingO. cumana races; (iii) identify nucleotide binding domain leucine rich repeat (NB-LRR) genes encoding R proteins conferring resistance to O. cumana in sunflower; (iv) increase the resolution of the chromosomal segment harboring Or₅ and related R genes through genetic and physical mapping in previously and newly developed mapping populations of sunflower; and (v) develop high-throughput DNA markers for rapidly and efficiently identifying and transferring sunflower R genes through marker-assisted selection. Revisions made during the course of project: Following changes in O. cumana race distribution in Israel, the newly arrived virulent race H was chosen for further analysis. HA412-HO, which was primarily chosen as a susceptible sunflower cultivar, was more resistant to the new parasite populations than var. Shemesh, thus we shifted sunflower research into analyzing the resistance of HA412-HO. We exceeded the deliverables for Objectives #3-5 by securing funding for complete physical and high-density genetic mapping of the sunflower genome, in addition to producing a complete draft sequence of the sunflower genome. We discovered limited diversity between the parents of the O. cumana population developed for the mapping study. Hence, the developed DNA marker resources were insufficient to support genetic map construction. This objective was beyond the scale and scope of the funding. This objective is challenging enough to be the entire focus of follow up studies. Background to the topic: O. cumana, an obligate parasitic weed, is one of the most economically important and damaging diseases of sunflower, causes significant yield losses in susceptible genotypes, and threatens production in Israel and many other countries. Breeding for resistance has been crucial for protecting sunflower from O. cumana, and problematic because new races of the pathogen continually emerge, necessitating discovery and deployment of new R genes. The process is challenging because of the uncertainty in identifying races in a genetically diverse parasite. Major conclusions, solutions, achievements: We developed a small collection of SSR markers for genetic mapping in O. cumana and completed a diversity study to lay the ground for objective #1. Because DNA sequencing and SNPgenotyping technology dramatically advanced during the course of the study, we recommend shifting future work to SNP discovery and mapping using array-based approaches, instead of SSR markers. We completed a pilot study using a 96-SNP array, but it was not large enough to support genetic mapping in O.cumana. The development of further SNPs was beyond the scope of the grant. However, the collection of SSR markers was ideal for genetic diversity analysis, which indicated that O. cumanapopulations in Israel considerably differ frompopulations in other Mediterranean countries. We supplied physical and genetic mapping resources for identifying R-genes in sunflower responsible for resistance to O. cumana. Several thousand mapped SNP markers and a complete draft of the sunflower genome sequence are powerful tools for identifying additional candidate genes and understanding the genomic architecture of O. cumana-resistanceanddisease-resistance genes. Implications: The OrobancheSSR markers have utility in sunflower breeding and genetics programs, as well as a tool for understanding the heterogeneity of races in the field and for geographically mapping of pathotypes.The segregating populations of both Orobanche and sunflower hybrids are now available for QTL analyses.
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Funkenstein, Bruria, and Cunming Duan. GH-IGF Axis in Sparus aurata: Possible Applications to Genetic Selection. United States Department of Agriculture, November 2000. http://dx.doi.org/10.32747/2000.7580665.bard.

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Many factors affect growth rate in fish: environmental, nutritional, genetics and endogenous (physiological) factors. Endogenous control of growth is very complex and many hormone systems are involved. Nevertheless, it is well accepted that growth hormone (GH) plays a major role in stimulating somatic growth. Although it is now clear that most, if not all, components of the GH-IGF axis exist in fish, we are still far from understanding how fish grow. In our project we used as the experimental system a marine fish, the gilthead sea bream (Sparus aurata), which inhabits lagoons along the Mediterranean and Atlantic coasts of Europe, and represents one of the most important fish species used in the mariculture industry in the Mediterranean region, including Israel. Production of Sparus is rapidly growing, however, in order for this production to stay competitive, the farming of this fish species has to intensify and become more efficient. One drawback, still, in Sparus extensive culture is that it grows relatively slow. In addition, it is now clear that growth and reproduction are physiological interrelated processes that affect each other. In particular sexual maturation (puberty) is known to be closely related to growth rate in fish as it is in mammals, indicating interactions between the somatotropic and gonadotropic axes. The goal of our project was to try to identify the rate-limiting components(s) in Sparus aurata GH-IGF system which might explain its slow growth by studying the ontogeny of growth-related genes: GH, GH receptor, IGF-I, IGF-II, IGF receptor, IGF-binding proteins (IGFBPs) and Pit-1 during early stages of development of Sparus aurata larvae from slow and fast growing lines. Our project was a continuation of a previous BARD project and could be divided into five major parts: i) obtaining additional tools to those obtained in the previous project that are necessary to carry out the developmental study; ii) the developmental expression of growth-related genes and their cellular localization; iii) tissue-specific expression and effect of GH on expression of growth-related genes; iv) possible relationship between GH gene structure, growth rate and genetic selection; v) the possible role of the IGF system in gonadal development. The major findings of our research can be summarized as follows: 1) The cDNAs (complete or partial) coding for Sparus IGFBP-2, GH receptor and Pit-1 were cloned. Sequence comparison reveals that the primary structure of IGFBP-2 protein is 43-49% identical to that of zebrafish and other vertebrates. Intensive efforts resulted in cloning a fragment of 138 nucleotides, coding for 46 amino acids in the proximal end of the intracellular domain of GH receptor. This is the first fish GH receptor cDNA that had been cloned to date. The cloned fragment will enable us to complete the GH - receptor cloning. 2) IGF-I, IGF-II, IGFBP-2, and IGF receptor transcripts were detected by RT-PCR method throughout development in unfertilized eggs, embryos, and larvae suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Preliminary RT-PCR analysis suggest that GH receptor transcript is present in post-hatching larvae already on day 1. 3) IGF-1R transcripts were detected in all tissues tested by RT-PCR with highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. Northern blot analysis detected IGF receptor only in gonads, brain and gill cartilage but not in muscle; GH increased slightly brain and gill cartilage IGF-1R mRNA levels. 4) IGFBP-2 transcript were detected only in liver and gonads, when analyzed by Northern blots; RT-PCR analysis revealed expression in all tissues studied, with the highest levels found in liver, skin, gonad and pyloric caeca. 5) Expression of IGF-I, IGF-II, IGF-1R and IGFBP-2 was analyzed during gonadal development. High levels of IGF-I and IGFBP-2 expression were found in bisexual young gonads, which decreased during gonadal development. Regardless of maturational stage, IGF-II levels were higher than those of IGF-L 6) The GH gene was cloned and its structure was characterized. It contains minisatellites of tandem repeats in the first and third introns that result in high level of genetic polymorphism. 7) Analysis of the presence of IGF-I and two types of IGF receptor by immunohistochemistry revealed tissue- and stage-specific expression during larval development. Immunohistochemistry also showed that IGF-I and its receptors are present in both testicular and ovarian cells. Although at this stage we are not able to pinpoint which is the rate-limiting step causing the slow growth of Sparus aurata, our project (together with the previous BARD) yielded a great number of experimental tools both DNA probes and antibodies that will enable further studies on the factors regulating growth in Sparus aurata. Our expression studies and cellular localization shed new light on the tissue and developmental expression of growth-related genes in fish.
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