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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2011 - 201813
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  • Publication
    Accès libre
    Nouveaux ligands du récepteur B₂ de la bradykinine : endocytose dirigée et catabolisme sélectif
    (2013) Roy, Caroline; Marceau, François
    Les récepteurs B₂ (RB₂) sont impliqués dans une multitude de réponses cellulaires notamment reliées à l’inflammation. Les RB₂ font partie du système kallikréine-kinine qui est en relation directe avec les composantes du système rénine-angiotensine. Une des composantes de ce dernier est l’enzyme de conversion de l’angiotensine (ECA) qui métabolise la bradykinine (BK) et la rend inactive. Un premier volet des travaux visait à étudier et caractériser de nouveaux agonistes du RB₂ qui sont prolongés en amino-terminal (N-terminal) ou conjugués à un épitope myc. Ces constructions révèlent de nouveaux outils d’imagerie cellulaire et permettent d’entrevoir la possibilité de pouvoir larguer un médicament conjugué aux ligands endogènes. Dans une seconde partie des travaux, un des agonistes prolongés en N-terminal, la cétirizine-ε-aminocaproyl-BK (CTZ-εACA-BK) et un peptide produit par le neutrophile humain (Met-Lys-BK-Ser-Ser) sont employés pour mettre à l’épreuve l’étendue du catabolisme de l’ECA. Seule Met-Lys-BK-Ser-Ser s’est révélée être un substrat fonctionnel de l’ECA.
  • Publication
    Accès libre
    Bifunctional epitope-agonist ligands of the bradykinin B2 receptor
    (De Gruyter, 2013-02-02) Gera, Lajos; Roy, Caroline; Marceau, François
    Two bradykinin (BK) B2 receptor agonists N-terminally extended with the myc epitope were synthesized and evaluated: myc-KPG-BK and myc-KGP-B-9972. The latter was modeled on the inactivation-resistant agonist B-9972 (D-Arg0, Hyp3, Igl5, Oic7, Igl8-BK) and is also resistant to endosomal inactivation. Despite a large loss of affinity relative to the parent peptide, the tagged analogs are conventional agonists in the umbilical vein contractility assay and compete for [3H]BK binding at the rabbit B2 receptor. Endocytosed myc-KGP-B-9972 most effectively carried AlexaFluor-488-conjugated anti-myc monoclonal antibodies into intact cells expressing the B2 receptor. Results support the prospects of functionally-active cargoes entering cells in a pharmacologically controlled manne
  • Publication
    Accès 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.
  • Publication
    Accès libre
    An in vitro reconstitution system to address the mechanism of the vascular expression of the bradykinin B1 receptor in response to angiotensin converting enzyme inhibition
    (Elsevier Science, 2011-10-02) Roy, Caroline; Marceau, Émilie; Gera, Lajos; Marceau, François
    The expression of the bradykinin (BK) B1 receptor (B1R), lacking in normal vascular tissues, is induced following innate immune system activation and chronic blockade of angiotensin converting enzyme (ACE). To identify cytokine-dependent or -independent mechanisms for the latter phenomenon, the ACE inhibitor enalaprilat and several peptides potentiated in vivo by ACE blockade were applied either directly to human umbilical artery smooth muscle cells (hUA-SMCs) or to differentiated monoblastoid U937 cells to produce a conditioned medium (CM) that was later transferred to hUA-SMCs. A phagocyte stimulant, lipopolysaccharide, did not upregulate B1R, measured using [3H]Lys-des-Arg9-BK binding, or translocate NF-κB to the nuclei if applied directly to the hUA-SMCs. However, the CM of lipopolysaccharide-stimulated U937 cells was active in these respects (effects inhibited by etanercept and correlated to TNF-α presence in the CM). A peptidase-resistant B1R agonist had no significant direct or indirect acute effect (4 h) on B1R expression, but repeated hUA-SMC stimulations over 40 h were stimulatory in the absence of NF-κB activation. Other peptides regulated by ACE or enalaprilat did not directly or indirectly stimulate B1R expression. The reconstitution system supports the rapid cytokine-dependent vascular induction of B1Rs and a slow “autoregulatory” one potentially relevant for the ACE blockade effect.
  • Publication
    Restreint
    Assessment of cation trapping by cellular acidic compartments
    (Academic Press, 2013-12-17) Roy, Caroline; Marceau, François; Bouthillier, Johanne; P. Michael, Conn
    All nucleated cells, from yeast to animal cells, concentrate cationic chemicals (weak bases with a pKa ~8-10) into acidic cell compartments (low retro-diffusion under a protonated form at low pH = ion trapping). The proton pump vacuolar (V)-ATPase is the driving force of this pseudotransport that concerns acidic organelles (mainly late endosomes and lysosomes). The latter rapidly become swollen (osmotic vacuolization) and macroautophagic. Cation concentration in cells is not proven to involve membrane transporters, but is prevented or reversed by inhibitors of V-ATPase, such as bafilomycin A1. Lipophilicity is a major determinant of the apparent affinity of this pseudotransport because simple diffusion of the uncharged form supports it. Quinacrine is a formerly used anti-parasitic drug that is intensely fluorescent, lipophilic and a tertiary amine. The drug, at micromolar concentrations, is proposed as a superior probe for assessing cation trapping by cellular acidic compartments, being readily quantified using fluorometry in cell extracts and analyzed using microscopy and cytofluorometry (fluorescence settings for fluorescein being applicable). Further, cells respond to micromolar levels of quinacrine by autophagic accumulation (e.g., accumulation of the activated macroautophagic effector LC3 II, immunoblots), an objective and universal response to sequestered amines.
  • Publication
    Accès libre
    Prolonged signalling and trafficking of the bradykinin B2 receptor stimulated with the amphibian peptide maximakinin : insight into the endosomal inactivation of kinins
    (Elsevier, 2011-11-15) Roy, Caroline; Bawolak, Marie-Thérèse; Gera, Lajos; Marceau, François
    Maximakinin, a 19-residue peptide from the amphibian Bombina maxima, incorporates the full sequence of bradykinin (BK) at its C-terminus with a hydrophilic 10-residue N-terminal extension. As a putative venom component, it may stimulate BK B2 receptors (B2Rs) in a distinct manner relative to the fragile mammalian agonist BK. Maximakinin affinity for B2Rs and angiotensin converting enzyme (ACE) and its pharmacological profile have been compared to those of BK. Maximakinin is an agonist of the human and rabbit B2R with a 8–12 fold lesser potency, but a prolonged duration of action relative to BK (ERK MAP kinase activation, c-Fos induction in HEK 293 cells). Maximakinin had a moderately inferior affinity (∼6-fold vs. BK) for recombinant ACE based on [3H]enalaprilat binding displacement. Unlike BK, maximakinin induced the internalization of the fusion protein B2R-green fluorescent protein (GFP) and the downregulation of this construction over a 12-h stimulation period, reproducing the effect of inactivation-resistant B2R agonists. Alternate homologues of BK extended at the N-terminus showed intermediate behaviours between BK and maximakinin in the B2R-GFP downregulation assay. The recycling of B2R-GFP at the cell surface after a 3-h BK treatment was notably inhibited by cotreatment with E-64 or bafilomycin A1, supporting that an endosomal cysteine protease degrades kinins in a process that determines the cycling and fate of the B2R. Maximakinin is the first known natural kinin sequence that elicits a prolonged cellular signalling, thus suggesting a possible basis for a venomous action and a naturally selected one for the design of B2R-transported biotechnological cargoes.
  • Publication
    Accès 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
  • Publication
    Accès 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.
  • Publication
    Accès 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.
  • Publication
    Accès libre
    High affinity capture and concentration of quinacrine in polymormonuclear neutrophils via vacuolar ATPase-mediated ion trapping : comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs
    (Elsevier, 2013-04-17) Roy, Caroline; Marceau, François; Fernandes, Maria J.; Gagné, Valérie
    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5 μM, ≥2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).