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Iorio-Morin C.,Université de Sherbrooke | Iorio-Morin C.,Institute Of Pharmacologie Of Sherbrooke | Germain P.,Université de Sherbrooke | Germain P.,Institute Of Pharmacologie Of Sherbrooke | And 8 more authors.
Cell Death and Differentiation | Year: 2012

Thromboxane A 2 (TXA 2) is an important lipid mediator whose function in apoptosis is the subject of conflicting reports. Here, a yeast two-hybrid screen for proteins that interact with the C-terminus of the TXA 2 receptor (TP) identified Siva1 as a new TP-interacting protein. Contradictory evidence suggests pro-and anti-apoptotic roles for Siva1. We show that a cisplatin treatment induces TXA 2 synthesis in HeLa cells. We demonstrate that endogenous TP stimulation promotes cisplatin-induced apoptosis of HeLa cells and that such modulation requires the expression of Siva1, as evidenced by inhibiting its endogenous expression using siRNAs. We reveal that, upon stimulation of TP, degradation of Siva1 is impeded, resulting in an accumulation of the protein, which translocates from the nucleus to the cytosol. Translocation of Siva1 correlates with its reduced interaction with Mdm2 (an inhibitor of p53 signalling), as well as with its increased interaction with TRAF2 and XIAP (known to enhance pro-apoptotic signalling). Our data provide a model that reconciles the pro-and anti-apoptotic roles that were reported for Siva1 and identify a new mechanism for promoting apoptosis by G protein-coupled receptors. Our findings may have implications in the use of cyclo-oxygenase inhibitors during cisplatin chemotherapy and might provide a target to reduce cisplatin toxicity on non-cancerous tissues. © 2012 Macmillan Publishers Limited All rights reserved.


Labrecque P.,Université de Sherbrooke | Labrecque P.,Institute Of Pharmacologie Of Sherbrooke | Roy S.J.,Université de Sherbrooke | Roy S.J.,Institute Of Pharmacologie Of Sherbrooke | And 8 more authors.
PLoS ONE | Year: 2013

Prostaglandin D2 (PGD2) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gαs-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gαi/o proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD2 induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1. © 2013 Labrecque et al.


Iorio-Morin C.,Université de Sherbrooke | Iorio-Morin C.,Institute Of Pharmacologie Of Sherbrooke | Germain P.,Université de Sherbrooke | Germain P.,Institute Of Pharmacologie Of Sherbrooke | And 2 more authors.
Electrophoresis | Year: 2013

Western blotting is a proven technique essential to a significant proportion of molecular biology projects. However, as results accumulate over the years, managing data can become daunting. Recognizing that the needs of a scientist working with Western blotting results are conceptually the same as those of a professional photographer managing a summer's worth of wedding photos, we report here a new workflow for managing Western blotting results using professional photo management software. The workflow involves (i) scanning all film-based results; (ii) importing the scans into the software; (iii) processing the scans; (iv) tagging the files with metadata, and (v) creating appropriate "smart-albums." Advantages of this system include space savings (both on our hard drives and on our desks), safer archival, quicker access, and easier sharing of the results. In addition, metadata-based workflows improve cross-experiment discovery and enable questions like "show me all blots labelled with antibody X" or "show me all experiments featuring protein Y". As project size and breadth increase, workflows delegating results management to the computer will become more and more important so that scientists can keep focussing on science. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Mathurin K.,Université de Sherbrooke | Mathurin K.,Institute Of Pharmacologie Of Sherbrooke | Gallant M.A.,Université de Sherbrooke | Gallant M.A.,Institute Of Pharmacologie Of Sherbrooke | And 14 more authors.
Journal of Biological Chemistry | Year: 2011

L-type prostaglandin synthase (L-PGDS) produces PGD2, a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD2-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2 -/--arr-3-/-. Arrestin-3 promotes PGD2 production by L-PGDS in vitro. IL-1β-induced PGD2 production is significantly lower in MEFs-arr-2-/--arr-3-/- than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD2 production in vitro and in MG-63 cells. We report the first characterization of an interactor/ modulator of a PGD2 synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Bourassa P.,University of Montréal | Bourassa P.,Université de Sherbrooke | Tudashki H.B.,University of Montréal | Pineyro G.,University of Montréal | And 6 more authors.
Molecular Pharmacology | Year: 2014

In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the μ-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2. Our results show that morphine and DAMGO display different profiles of receptor internalization and a similar ability to trigger the phosphorylation of ERK1/ERK2. Our SPR analyses revealed that morphine and DAMGO evoke similar SPR signatures and that Gαi, cAMP-dependent pathways, and ERK1/ERK2 have key roles in morphine- and DAMGO-mediated signaling. Most interestingly, we found that the so-called MOP neutral antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), naloxone, and naltrexone behave like partial agonists. Even more intriguing, BRET experiments indicate that CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2) induces similar conformational changes as naltrexone at the Gαi-βγ interface, whereas it appears as an inverse agonist based on its SPR response thus indicating distinct signaling mechanisms for the two ligands. Taken together, our results support the usefulness of label-free methods such as SPR to study whole-cell responses and signaling cascades triggered by G protein-coupled receptors and complement the conventional approaches by revealing cellular responses that would have been otherwise undetectable. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.


Wardman J.H.,Yeshiva University | Zhang X.,Yeshiva University | Gagnon S.,Institute Of Pharmacologie Of Sherbrooke | Castro L.M.,University of Sao Paulo | And 4 more authors.
Journal of Neurochemistry | Year: 2010

Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, ∼1/3 were decreased in the PC1/3 null mice relative to wild-type mice, ∼1/3 showed no change, and ∼1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC. © 2010 International Society for Neurochemistry.


Lachance V.,Université de Sherbrooke | Lachance V.,Institute Of Pharmacologie Of Sherbrooke | Degrandmaison J.,Université de Sherbrooke | Degrandmaison J.,Institute Of Pharmacologie Of Sherbrooke | And 10 more authors.
Journal of Cell Science | Year: 2014

We and others have shown that trafficking of G-protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Here, we show by overexpression and depletion studies that HACE1, a HECTdomain-containing ubiquitin ligase, promotes the recycling of the b2-adrenergic receptor (β2AR), a prototypical G-protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the β2AR in conjunction with HACE1 triggered ubiquitylation of Rab11a, as observed by western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitylated on Lys145. A Rab11a-K145R mutant failed to undergo β2AR-HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the β2AR. Rab11a, but not Rab11a-K145R, was activated by β2AR- HACE1, indicating that ubiquitylation of Lys145 is involved in activation of Rab11a. Co-expression of β2AR-HACE1 also potentiated ubiquitylation of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases through their ubiquitylation, with associated functional effects demonstrated on Rab11a. This suggests a new pathway whereby a cargo protein, such as a Gprotein-coupled receptor, can regulate its own trafficking by inducing the ubiquitylation and activation of a Rab GTPase. © 2014. Published by The Company of Biologists Ltd.


Murza A.,Université de Sherbrooke | Murza A.,Institute Of Pharmacologie Of Sherbrooke | Sainsily X.,Université de Sherbrooke | Sainsily X.,Institute Of Pharmacologie Of Sherbrooke | And 21 more authors.
Journal of Medicinal Chemistry | Year: 2016

ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELA's structure-activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the Gαi1 and β-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases. © 2016 American Chemical Society.


Tremblay H.,Institute Of Pharmacologie Of Sherbrooke | St-Georges C.,Institute Of Pharmacologie Of Sherbrooke | Legault M.-A.,Institute Of Pharmacologie Of Sherbrooke | Morin C.,SCF Pharma | And 2 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2014

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells. © 2014 Elsevier Ltd.

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