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Personne :
Roy, Caroline

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Roy

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Caroline

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Université Laval. Département de microbiologie-infectiologie et d'immunologie

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ncf13705806

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Voici les éléments 1 - 5 sur 5
  • PublicationAccès libre
    Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling
    (Academic Press, 2013-05-01) Fortin, Sébastien; Roy, Caroline; Marceau, François; Lodge, Robert; Gera, Lajos; C. Gaudreault, René.; Charest-Morin, Xavier
    The bradykinin (BK) B2 receptor (B2R) is G protein coupled and phosphorylated upon agonist stimulation; its endocytosis and recycling are documented. We assessed the effect of drugs that affect the cytoskeleton on B2R cycling. These drugs were targeted to tubulin (paclitaxel, or the novel combretastatin A-4 mimetic 3,4,5-trimethoxyphenyl-4-(2-oxoimidazolidin-1-yl)benzenesulfonate [IMZ-602]) and actin (cytochalasin D). Tubulin ligands did not alter agonist-induced receptor endocytosis, as shown using antibodies reactive with myc-tagged B2Rs (microscopy, cytofluorometry), but rather reduced the progression of the ligand–receptor–β-arrestin complex from the cell periphery to the interior. The 3 fluorescent probes of this complex (B2R-green fluorescent protein [B2R-GFP], the fluorescent agonist fluorescein-5-thiocarbamoyl-D-Arg-[Hyp3, Igl5, Oic7, Igl8]-BK and β-arrestin2–GFP) were condensed in punctuate structures that remained close to the cell surface in the presence of IMZ-602. Cytochalasin D selectively inhibited the recycling of endocytosed B2R-GFP (B2R-GFP imaging, [3H]BK binding). Dominant negative (GDP-locked)-Rab5 and -Rab11 reproduced the effects of inhibitors of tubulin and actin, respectively, on the cycling of B2R-GFP. GDP-locked-Rab4 also inhibited B2R-GFP recycling to the cell surface. Consistent with the displacement of cargo along specific cytoskeletal elements, Rab5-associated progression of the endocytosed BK B2R follows microtubules toward their (−) end, while its recycling progresses along actin fibers to the cell surface. However, tubulin ligands do not suppress the tested desensitization or resensitization mechanisms of the B2R
  • PublicationAccès libre
    Bifunctional ligands of the bradykinin B2 and B1 receptors : an exercise in peptide hormone plasticity
    (Elsevier, 2018-05-23) Bachelard, Hélène; Roy, Caroline; Bawolak, Marie-Thérèse; Fortin, Jean-Philippe.; Marceau, François; Morissette, Guillaume; Lajos, Gera; Charest-Morin, Xavier
    Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B2 receptor (B2R) and to the inducible B1 receptor (B1R). B2Rs and B1Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B2Rs, Lys-des-Arg9-BK for B1R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B2R antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at B2R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value.
  • PublicationAccès libre
    Bradykinin receptors : agonists, antagonists, expression, signaling and adaptation to sustained stimulation
    (2013-12-01) Fortin, Sébastien; Sabourin, Thierry; Roy, Caroline; Bawolak, Marie-Thérèse; Koumbadinga, Gérémy Abdull; Fortin, Jean-Philippe.; Marceau, François; Morissette, Guillaume; Lodge, Robert; C. Gaudreault, René.; Houle, Steeve.; Charest-Morin, Xavier; Bouthillier, Johanne; Gera, Lajos
    Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein–coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and nonpeptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. Both receptor subtypes mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors. While both receptor subtypes are mainly coupled to the Gq protein and related second messengers, the B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation.
  • PublicationAccès libre
    N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes : structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes
    (Elsevier, 2012-02-18) Gera, Lajos; Roy, Caroline; Bawolak, Marie-Thérèse; Marceau, François; Charest-Morin, Xavier
    Peptide agonists and antagonists of both bradykinin (BK) B1 and B2 receptors (B1R, B2R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ɛ-aminocaproyl (ɛ-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B1R: B-10376: CF-ɛ-ACA-Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-BK; B2R: B-10380: CF-ɛ-ACA-d-Arg-[Hyp3, Igl5, d-Igl7, Oic8]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B2R agonists CF-ɛ-ACA-BK, AF350-ɛ-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B1R agonist B-10378 (CF-ɛ-ACA-Lys-des-Arg9-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ɛ-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.
  • PublicationAccès libre
    Pharmacological profile of a bifunctional ligand of the formyl peptide receptor1 fused to the myc epitope
    (Elsevier, 2015-02-10) Roy, Caroline; Marceau, François; Fernandes, Maria J.; Charest-Morin, Xavier
    In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10–1000 nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~ 7 nM (microscopy). The nonfluorescent version of the antibody (28 nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc.