Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein–coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal–regulated kinase 1/2 mitogen-activated protein kinases through the Gi class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2

Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs

La protéine régulatrice de la glucokinase hépatique: études cinétiques et clonage de l'ADNc

Doctorat en sciences agronomiques -- UCL, 199

Deorphanization of G-protein-coupled receptors.

G-protein-coupled receptors constitute one of the major families of drug targets. Orphan receptors, for which the ligands and function are still unknown, are an attractive set of future targets for presently unmet medical needs. Screening strategies have been developed over the years in order to identify the natural ligands of these receptors. Natural or chimeric G-proteins that can redirect the natural coupling of receptors toward intracellular calcium release are frequently used. Potential problems include poor expression or trafficking to the cell surface, constitutive activity of the receptors, or the presence of endogenous receptors in the cell types used for functional expression, leading to nonspecific responses. Many orphan receptors characterized over the last 10 years have been associated with previously known bioactive molecules. However, new and unpredicted biological mediators have also been purified from complex biological sources. A few old and recent examples, including nociceptin, chemerin, and the F2L peptide are illustrated. Future challenges for the functional characterization of the remaining orphan receptors include the potential requirement of specific proteins necessary for quality control, trafficking or coupling of specific receptors, the possible formation of obligate heterodimers, and the possibility that some constitutively active receptors may lack ligands or respond only to inverse agonists. Adapted expression and screening strategies will be needed to deal with these issues.Journal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: re.jinfo:eu-repo/semantics/publishe

Heterologous expression of an active rat regulatory protein of glucokinase

The cDNA presumed to encode the rat liver regulatory protein of glucokinase has been expressed in Escherichia coli and a partially soluble protein has been obtained. This recombinant protein was partially purified and found to have the same apparent molecular mass as the regulatory protein purified from rat liver. Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate

Effect of sulphur mustard on the expression of urokinase in cultured 3T3 fibroblasts.

The expression of plasminogen activator (PA), a serine proteinase involved in the degradation of extracellular matrix proteins, has been investigated in 3T3 fibroblasts after in vitro exposure to sulphur mustard (SM). Expression of the cell-associated enzyme has been assessed with a synthetic substrate assay and at the mRNA level. Twenty-four hours after 100 microM SM, cell viability (monitored by MTT assay) was not significantly affected, but protein synthesis (tritiated leucine incorporation) was reduced to < 20% of the control value. PA activity was significantly increased compared to control cells with a 20-fold increase after 24 h. This up-regulation was independent of the cell density, occurred maximally between days 1 and 4 and persisted for at least 6 days after exposure. Lower concentrations of SM (< or = 10 microM) did not significantly affect PA activity. Northern blotting experiments revealed an increased expression of urokinase (u-PA) transcripts in cells treated with 100 microM SM, with a peak at 10 h after exposure. Conditioned culture medium from cell cultures treated with 100 microM SM did not affect the expression of PA activity in naive or SM-treated cultures. Thiodiglycol (100 microM), the main metabolite of SM, did not influence the expression of PA in the same system. Different compounds were tested for modulation of the PA upregulation after SM exposure. Nicotinamide (5 mM), vitamin D3 (10(-10)M), extracellular calcium (2 mM) or EGTA (5 mM) had no effect. Ryanodine (10 microM) amplified the PA up-regulation by a factor of 2 and vanadate (500 microM) reduced it by approximately 50%. Dexamethasone (1 microM) added directly after SM treatment almost completely prevented the induction of PA at both the protein and mRNA levels. Overall these results demonstrate an up-regulation of urokinase in 3T3 fibroblasts after treatment with SM, which is possibly mediated by intracellular calcium mobilization. Further studies are needed to identify the significance of this proteolytic response in the pathogenesis of blistering and/or DNA repair mechanisms.Comparative StudyJournal Articleinfo:eu-repo/semantics/publishe

Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation.

In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6-phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1-phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half-maximal effect increased linearly with the concentrations of sorbitol 6-phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase. Sorbitol 6-phosphate, 2-deoxysorbitol 6-phosphate and mannitol 1-phosphate, all analogs of the open-chain configuration of fructose 6-phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6-phosphate, whereas fixed analogs of the furanose form of fructose 6-phosphate were inactive or behaved as activators. This indicated that fructose 6-phosphate in its open-chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1-phosphate, psicose 1-phosphate, ribitol 5-phosphate, analogs of fructose 1-phosphate and of ribitol 5-phosphate and, at much higher concentrations, inorganic phosphate

[A New Mechanism for the Short-term Regulation of Liver Glucokinase]

Fructose stimulates the phosphorylation of glucose in the liver. The mechanism of this effect involves a regulatory protein, which, in the presence of fructose 6-phosphate, binds to, and inhibits glucokinase. Fructose gives rise to fructose 1-phosphate, which binds to the regulatory protein and causes its dissociation from glucokinase, thus allowing this enzyme to be fully active. Because of this effect, fructose can play the role of a signal which tells the liver that glucose is absorbed from the gut