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

ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23−/− mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23−/− mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies

Etude de la relation structure/fonction de l'enzyme de synthèse de l'inositol 1,3,4,5- tétrakisphosphate :l'inositol 1,4,5- trisphosphate 3-kinase

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Etude de la relation structure/fonction de l'enzyme de synthèse de l'inositol 1,3,4,5- tétrakisphosphate :l'inositol 1,4,5- trisphosphate 3-kinase

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Identification of an active site cysteine residue in human type I Ins(1,4,5)P3 5-phosphatase by chemical modification and site-directed mutagenesis

Chemical modification using thiol-directed agents and site-directed mutagenesis have been used to investigate the crucial role of an active site cysteine residue within the substrate-binding domain of human type I Ins(1,4,5)P3 5-phosphatase. Irreversible inhibition of enzymic activity is provoked by chemical modification of the enzyme by N-ethylmaleimide (NEM), 5,5'-dithio-2-nitrobenzoic acid, iodoacetate and to a much smaller extent by iodoacetämide. The alkylation reaction by NEM is prevented in the presence of Ins(1,4,5)P3. The results indicate that NEM binds at the active site of the enzyme with a stoichiometry of 0.9 mol of NEM per mol of enzyme. A single [14C]NEM-modified peptide was isolated after alpha-chymotrypsin proteolysis of the radiolabelled enzyme and reverse-phase HPLC. Sequence analysis of the active site-labelled peptide (i.e. MNTRCPAWCD) demonstrated that Cys348 contained the radiolabel. Furthermore two mutant enzymes were obtained by site-directed mutagenesis of the cysteine residue to serine and alanine respectively. Both mutant enzymes had identical UV CD spectra. The two mutants (i.e. Cys348-->Ser and Cys348-->Ala) show a marked loss of enzymic activity (more than 98% compared with the wild-type enzyme). Thus we have directly identified a reactive cysteine residue as part of the active site, i.e. the substrate-binding domain, of Ins(1,4,5)P3 5-phosphatase. This cysteine residue is part of a sequence 10 amino acids long that is well conserved among the primary structures of inositol and phosphatidylinositol polyphosphate 5-phosphatases.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Identification of Three Active Site Residues Involved in Substrate Binding by Human 43 kDa-D-myo-Inositol, 1,4,5-Trisphosphate 5-Phosphatase

info:eu-repo/semantics/publishe

Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses

The formyl peptide receptor (FPR) family is involved in host defence against pathogens, but also in sensing internal molecules that may constitute signals of cellular dysfunction. It includes three subtypes in human and other primates. FPR responds to formyl peptides derived from bacterial and mitochondrial proteins. FPRL1 displays a large array of exogenous and endogenous ligands, including the chemokine variant sCKbeta8-1, the neuroprotective peptide humanin, and lipoxin A4. Two high affinity agonists (F2L and humanin) were recently described for FPRL2. In mouse, eight FPR-related receptors have been described. Fpr1 is the ortholog of human FPR, while fpr2 appears to share many ligands with human FPRL1. Altogether, the physiological role of the FPR family is still incompletely understood, due in part to the large variety of ligands, the redundancy with other chemoattractant agents, and the lack of clear orthologs between human and mouse receptors. Newly developed tools will allow to study further this family of receptors.Journal ArticleResearch Support, Non-U.S. Gov'tReviewinfo:eu-repo/semantics/publishe

Calcium-Calmodulin-dependent protein kinase II and protein kinase C-mediated phosphorylation and activation of D-myo-inositol 1,4,5-trisphosphate 3-kinase B in astrocytes

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase catalyzes the production of D-myo-inositol 1,3,4,5-tetrakisphosphate from the second messenger Ins (1,4,5)P3. Transient and okadaic acid-sensitive activation of Ins(1,4,5)P3 3-kinase by 8-10-fold is observed in homogenates prepared from rat cortical astrocytes after incubation with either carbachol or UTP. 12-O-Tetradecanoylphorbol-13-acetate provokes the activation of Ins(1,4,5)P3 3-kinase by 2-fold in both cell systems. The kinase was purified by calmodulin-Sepharose from the two cell systems. Enzyme activity corresponding to the silver-stained 88-kDa protein could be regenerated after SDS-polyacrylamide gel electrophoresis. Antibodies to two distinct peptides chosen in the primary structure of human Ins(1,4,5)P3 3-kinase B recognized the astrocytic native isoform. In [32P]orthophosphate-preincubated cells, a major phosphorylated 88-kDa enzyme could be purified and identified in cells in response to receptor activation or 12-O-tetradecanoylphorbol-13-acetate treatment. Calmodulin kinase II inhibitors (i.e. KN-93 and KN-62) and a protein kinase C inhibitor (i.e. calphostin C) prevented the phosphorylation of the 88-kDa isoenzyme. In addition to enzyme activation, a redistribution of Ins(1,4,5)P3 3-kinase from soluble to particulate fraction of astrocytes was observed. In vitro phosphorylation of the purified enzyme by calmodulin kinase II and protein kinase C added together resulted in a maximal 60-70-fold activation.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe