Identification of three distinct receptor binding sites of murine interleukin-11.
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Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130. (+info)
Adhesion energy of receptor-mediated interaction measured by elastic deformation.
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We investigated the role of receptor binding affinity in surface adhesion. A sensitive technique was developed to measure the surface energy of receptor-mediated adhesion. The experimental system involved a functionalized elastic agarose bead resting on a functionalized glass coverslip. Attractive intersurface forces pulled the two surfaces together, deforming the bead to produce an enlarged contact area. The Johnson-Kendall-Roberts (JKR) model was used to relate the surface energy of the interaction to the elasticity of the bead and the area of contact. The surface energies for different combinations of modified surfaces in solution were obtained from reflection interference contrast microscopy (RICM) measurements of the contact area formed by the bead and the coverslip. Studies with surfaces functionalized with ligand-receptor pairs showed that the relationship between surface energy and the association constant of the ligand binding has two regimes. At low binding affinity, surface energy increased linearly with the association constant, while surface energy increased logarithmically with the association constant in the high affinity regime. (+info)
Molecular biology of biotin attachment to proteins.
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Enzymatic attachment of biotin to proteins requires the interaction of a distinct domain of the acceptor protein (the "biotin domain") with the enzyme, biotin protein ligase, that catalyzes this essential and rare post-translational modification. Both biotin domains and biotin protein ligases are very strongly conserved throughout biology. This review concerns the protein structures and mechanisms involved in the covalent attachment of biotin to proteins. (+info)
Human biotinidase isn't just for recycling biotin.
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For years, the major role of biotin has been as the coenzyme for four carboxylases in humans. Although there has been evidence that biotin might have other functions, none has been firmly established. The discovery that human serum biotinidase has biotinyl-transferase activity, in addition to biotinidase hydrolase activity, presents new possibilities for the role of biotinidase in biotin metabolism. Specific transfer of biotin to histones by biotinidase provides a possible explanation for why biotin is found in the nucleus and the nature of its role in the regulation of protein transcription. Future studies will help to determine the functions of biotinidase in biotin metabolism and in disease states. (+info)
Cellular uptake of biotin: mechanisms and regulation.
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This review describes our knowledge of biotin transport in the small intestine of humans and other mammals and presents recent findings in the area. Previous studies have shown that biotin transport across the brush border membrane of the small intestinal absorptive cells occurs via a carrier-mediated, Na+ gradient-dependent, electroneutral mechanism. Exit of biotin out of the enterocyte, i.e., transport across the basolateral membrane, also occurs via a carrier-mediated process, but the process is Na+ independent and electrogenic. Recent studies from our laboratory have shown that the uptake process of biotin in Caco-2 cells, a human-derived cultured intestinal epithelial cell line, are under the cellular regulation of both a protein kinase C- and a Ca/calmodulin-mediated pathway. In addition, the uptake process is shared by another water-soluble vitamin, pantothenic acid. For the first time, other recent studies have detected the existence of a Na+-dependent, carrier-mediated mechanism for biotin uptake at the apical membrane of colonocytes, which could theoretically mediate absorption of the biotin synthesized by colonic microflora. This system was again found to be shared by pantothenic acid, which is also synthesized by the normal microflora of the large intestine. (+info)
Advanced analysis of biotin metabolites in body fluids allows a more accurate measurement of biotin bioavailability and metabolism in humans.
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In previous studies, the bioavailability of biotin in humans was estimated from the recovery of biotin in urine; urinary biotin was measured by microbial growth assays or assays of avidin-binding activity. These assays underestimate concentrations of biotin metabolites, which originate from beta-oxidation, sulfur oxidation or a combination. We have developed an HPLC/avidin-binding assay that is specific for biotin and its metabolites. With the use of the HPLC/avidin-binding assay, TLC and derivatization with p-dimethylaminocinnamaldehyde, we have identified and quantitated biotin and metabolites in urine from six healthy adults. Of that total, biotin accounted for 32+/-12%, bisnorbiotin for 52+/-15%, bisnorbiotin methyl ketone for 7.9+/-5.8%, biotin-d,l-sulfoxide for 4.0+/-3.2% and biotin sulfone for 3.6+/-1.9%. After intravenous administration of 18.4 micromol of biotin, the urinary excretion of biotin metabolites increased 21-130 times above baseline values. Because the biliary excretion of biotin is quantitatively minor (1.9+/-0.2% of an intravenous [14C]biotin dose in rats), intravenously administered biotin is not exposed to intestinal microorganisms. Thus we conclude that biotin metabolites in human urine originate from biotin catabolism in human tissues rather than biotin catabolism by intestinal microorganisms. With the use of the HPLC/avidin-binding assay, we estimated the bioavailability of biotin in adults from the urinary excretion of biotin and metabolites after ingestion of 2.1, 8.2 and 81.9 micromol of biotin. These data provide evidence that biotin is nearly completely absorbed. (+info)
Biotin status: which are valid indicators and how do we know?
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Although estimated average requirements for biotin have been proposed, the human requirements for biotin in specific populations and at various ages remain uncertain, in part because indicators of biotin status have not been validated. With the use of improved methods for measuring biotin and metabolites, a recent study indicated that decreased urinary excretion of biotin and bisnorbiotin is an early and sensitive indicator of biotin deficiency, but decreased serum concentration of biotin is not. Increased urinary excretion of 3-hydroxyisovaleric acid (3-HIA), a leucine metabolite that is excreted in increased quantities with deficiency of the biotin-dependent enzyme beta-methylcrotonyl-CoA carboxylase, is also an early and sensitive indicator of biotin deficiency. When these indicators were assessed longitudinally in 13 pregnant women, biotin excretion was not significantly decreased early in pregnancy but did decrease significantly from early to late pregnancy. Excretion of 3-HIA was abnormally increased in about three-fourths of the women studied in both early and late pregnancy. Thus, each indicator detected biotin deficiency late in pregnancy, but assessment of biotin status for the two indicators conflicted early in pregnancy. Preliminary results from a trial assessing response of 3-HIA excretion to biotin treatment indicate that biotin status is indeed impaired both early and late in pregnancy. (+info)
Mapping binding domains of kininogens on endothelial cell cytokeratin 1.
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Human cytokeratin 1 (CK1) in human umbilical vein endothelial cells (HUVEC) is expressed on their membranes and is able to bind high molecular weight kininogen (HK) (Hasan, A. A. K., Zisman, T., and Schmaier, A. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3615-3620). New investigations have been performed to demonstrate the HK binding domain on CK1. Four overlapping recombinant (r) CK1 proteins were produced in Escherichia coli by a glutathione S-transferase gene fusion system. Biotin-HK specifically bound to rCK128 and rCK131 in the presence of Zn2+ but not to Deleted1-6rCK131. Recombinant CK128 and rCK131 also inhibited biotin-HK binding to HUVEC with IC50 of 0.4 and 0.5 microM, respectively. Alternatively, rCK114 and Deleted1-6rCK131 did not inhibit binding at concentrations >/=1 microM. Seven sequential 20 amino acid peptides of CK1 were prepared to cover the protein coded by exons 1-3. Only the first peptide (GYG20) coded by exon 1 significantly inhibited HK binding to HUVEC with an IC50 of 35 microM. Fine mapping studies isolated two overlapping peptides also coded by exon 1 (GPV15 and PGG15) that inhibited binding to HUVEC with IC50 of 18 and 9 microM, respectively. A sequence scrambled peptide of PGG15 did not block binding to HUVEC and biotin-GPV20 specifically bound to HK. Peptides GPV15 and PGG15 also blocked prekallikrein activation on endothelial cells. However, inhibition of PK activation by peptide PGG15 occurred at 10-fold lower concentration (IC50 = 1 microM) than inhibition of biotin-HK binding to HUVEC (IC50 = 10 microM). These studies indicate that HK binds to a region of 20 amino acids coded by exon 1 on CK1 which is carboxyl-terminal to its glycine-rich amino-terminal globular domain. Furthermore, HK binding to CK1 modulates PK activation on HUVEC. (+info)