DR PLOUFFE'S PATH TOWARD HER ACTUAL RESEARCH PROGRAMME

Dr Plouffe completed biochemistry undergraduate studies at Université de Sherbrooke (QC, Canada) (2000-2003). Then, she quickly developed a marked interest for G protein-coupled receptor (GPCR) signalling. As GPCRs represent the largest family of cell surface proteins, and control a vast repertoire of physiological functions, these receptors are the actual target of 30-50% of all prescribed drugs. She started her journey in this exciting research field by investigating the molecular mechanisms involved in the angiotensin type 2 receptor (AT₂R)-mediated neurite outgrowth as a MSc student (2003-2005). This work contributed to the understanding of the mechanisms underlying neuronal differentiation required for brain development. During her MSc studies, Dr Plouffe also contributed to the characterisation of new selective AT₂R agonists in collaboration with research teams from Uppsala University (Sweden). Then, Dr Plouffe completed a PhD in Neuroscience at University of Ottawa (ON, Canada) (2006-2011), where she identified the molecular mechanisms involved in the opposite regulation of dopamine D1 and D5 receptors by protein kinase C. Her work shed light on the mechanisms underlying the positive action of prefrontal cortex on motor activity and of the thalamic parafascicular nucleus on awareness. 

Dr Plouffe decided to pursue her research in the field of GPCR signalling by completing a postdoctoral training at the Institute for Research in Immunology and Cancer, part of University of Montreal (QC, Canada) (2012-2018) as part of Prof Michel Bouvier's research team, a world-wide recognised expert in the field of GPCR signalling. During her postdoctoral training, Dr Plouffe used Bioluminescence Resonance Energy Transfer (BRET)-based technology to tackle important questions related to GPCRs involved in metabolic disorders. In collaboration with Prof Ralf Jockers from Institut Cochin (France), she investigated the signalling parameters of the melatonin type 2 receptor preventing development of type 2 diabetes. She also worked on the ghrelin receptor, involved in energy homeostasis and food intake, in collaboration with Prof Birgitte Holst (University of Copenhagen). This study lead to the identification of the ghrelin receptor-induced biased signalling mediated by a new compound responsible for unique physiological outcomes observed in murine model. This was one of the rare studies translating biased signalling from a receptor to in vivo functions.

Dr Plouffe had also the joy to participate to a jointed collaboration with Prof Robert J. Lefkowitz, a Nobel Laureate from Duke University (USA), and Prof Georgios Skiniotis, an expert in electron cryomicroscopy (cryoEM) from Stanford University (USA) on a project that was particularly inspiring and determinant for her actual research programme. Classically, G protein activation by GPCRs occurs at the plasma membrane and is rapidly terminated by the recruitment of a protein called β-arrestin to the activated GPCRs. This promotes G protein uncoupling from receptor, GPCR internalisation and signalling arrest. However, during the past decade, emerging evidence suggest that many GPCRs can continue to activate G proteins from internal compartments after they have been internalized. This fruitful collaboration allowed to appreciate the important role of β-arrestin in this novel type of G protein signalling and provided strong evidence for the formation of signalling complexes (called megaplexes) composed of the GPCR, G proteins and β-arrestin.

DR PLOUFFE'S RESEARCH PROGRAMME

The spatiotemporal properties of GPCRs signalling in intracellular compartments are different than those of GPCRs signalling at the plasma membrane. The absence of desensitising effect of β-arrestin on GPCRs signalling in intracellular compartments is generally translated to a sustained and prolonged signalling as opposed to the transient kinetic observed for GPCRs signalling at the plasma membrane. Furthermore, G protein signalling in intracellular compartments such as endosomes and Golgi allows a proximity with the nucleus, which favours the modulation of transcription factors, leading to changes in gene expression. As a newly appointed Vice-Chancellor’s Fellow and principal investigator at Queen’s University Belfast, Dr Plouffe aims to tackle important mechanistic questions underlying this novel signalling mode. Importantly, she wants to take advantage of the different spatiotemporal properties of compartmentalised G protein signalling to design new pharmacological approaches to fight several diseases. Dr Plouffe's current targets are type 2 diabetes, glioblastoma multiforme, melanoma, and atherosclerosis.

CURRENT PROJECTS IN THE LAB

1- To answer the following mechanistic questions concerning compartmentalised G protein signalling and megaplex formation:

• Is G protein selectivity different at intracellular compartments vs. at plasma membrane?
• Is megaplex formation restricted to Gαs isoform?
• What is the specific role of β-arrestin within the megaplex?

2- To investigate the role of endosomal G protein signalling by the viral chemokine GPCR US28 in glioblastoma multiforme (GBM) malignancy using glioblastoma cells (U251).

• Determine the constitutive Gαq protein spatial activation profile of US28 in these cells.
• Determine the effect of reduced US28 constitutive internalisation on this spatial activation profile.
• Evaluate the role of endosomal US28 signalling in proliferation, invasiveness, angiogenesis, and inflammation in these cells. 
• Use of pH-sensitive nanoparticles to deliver drugs or nanobodies directly into endosomes to block US28 and characterisation of these encapsulated drugs on US28-induced signalling and GBM malignancy.

3- To investigate compartmentalised G protein signalling by the chemokine GPCR CCR7, involved in melanoma by comparing 2 biased agonists (CCL19 and CCL21) for their respective ability to induce:

• β-arrestin recruitment to CCR7
• Internalisation of CCR7
• Gαi activation by CCR7 at the plasma membrane and in endosomes

4- To investigate the role of endosomal Gαs activation in GLP1R-induced insulin secretion by rat insulinoma INS-1 832/3 cells.

• Design and physically characterise pH-sensitive mesoporous silica nanoparticles (MSNs) cloaked with a pegylated liposome covalently linked to exendin-4 (ex4-MSNs) loaded with exendin-4 (ex4-MSN-ex4) or empty (ex4-MSN-Φ).
• Measurement of MSNs uptakes by INS-1 832/3 cells in presence or absence of internalisation inhibitors.
• Measurement of the following GLP1R-mediated Gs signalling at plasma membrane and in early endosomes upon stimulation with exendin-4, ex4-MSN-ex4, or ex4-MSN-Φ.
• Measurement of insulin secretion by INS-1 832/3 cells upon stimulation with exendin-4, ex4-MSN-ex4, or ex4-MSN-Φ.

GROUP MEMBERS

Technicians:

• Mr Shane Houston 2019-Present

PhD Students:

• Miss Carole Daly 2019-Present

Project: Non-canonical G protein signalling of US28 as a new target for glioblastoma.

MSc Students:

• Mr Stephen Copeland 2019-2020

Project: Non-canonical G protein signalling of US28 as a new target for glioblastoma.

Undergraduate Students:

• Mr Adam McShane 2019-2020

Summer Students:

• Miss Éloise Heulet 2019 (Advanced Biology, Université de Nantes, France)
• Miss Lenka Toderova 2019 (Nuffield Research Placement)
• Mr Cole Roberts 2019 (Medical School, University of Dundee, UK)

COLLABORATORS

 National:

• Prof David Grieve (WWIEM, Queen's University Belfast)
• Dr Emma Evergren Mills (CCRCB, Queen's University Belfast)
• Dr Irina Tikhonova (School of Pharmacy, Queen's University Belfast)
• Prof Qingbo Xu (King's College London)
• Dr Alejandra Tomas (Imperial College London)
• Prof I. Sadaf Farooqi (Wellcome-MRC Institute of Metabolic Science, University of Cambridge) 

 International:

• Prof Martine J. Smit (Vrije Universiteit Amsterdam, Netherlands)
• Prof Nigel W. Bunnett (New York University, USA)
• Dr Alex Thomsen (New York University, USA)
• Dr Arun K. Shukla (Indian Institute of Technology Kanpur, India) 

SELECTED AWARDS

• 2018 Wellcome Trust Seed Award in Science
• 2017 Travel Award from the Institute for Research in Immunologie and Cancer
• 2016 Étudiant-Chercheurs Étoiles Award from Fonds de la Recherche du Québec en Santé
• 2016 Diabetes Canada Postdoctoral Award
• 2012 Canadian Institutes for Health Research Postdoctoral Award
• 2010 Best poster Award at the 2nd Brain Health and Research Day (Ottawa, Canada)
• 2010 University of Ottawa Tuition Fee Schorlarship
• 2009 Best poster Award at the 1^st Brain Health and Research Day (Ottawa, Canada)
• 2008 Travel Award from the American Society for Biochemistry & Molecular Biology
• 2007 Fonds de la Recherche du Québec en Santé Doctoral Award
• 2007 University of Ottawa Excellence Scholarship
• 2005 Best Poster Award from the Société de Neuroendocrinologie (France)
• 2004 Best Oral Presentation Award from Équipe de Physiopathologie Endocrinienne (Qc, Canada)

INVITED SPEAKER

• 2019 Invited Short Talk - Trafficking in Cancer Biology: Interplay, Targeting and Therapy (October 21-24, Turin - Italy).
• 2018 Invited speaker - Seminar Series, Department of Pharmaceutical Chemistry, Philipps-Universität Marburg (June 12^th, Marburg - Germany).
• 2018 Invited speaker - Seminar Series, Medical School, University of Nottingham (June 4^th, Nottingham - UK).
• 2018 Invited speaker - Seminar Series, Department of Medicinal Chemistry, Virginia Commonwealth University (May 24^th, Richmond - USA).
• 2017 Speaker - Seminar Series, Institute for Research in Immunology and Cancer, University of Montreal (September 22^nd, Montreal - Canada).
• 2017 Speaker - 7th Scientific Day, Institute for Research in Immunology and Cancer, University of Montreal (May 26^th, Montreal - Canada).
• 2017 Selected Short Talk - Molecular Pharmacology Gordon Research Seminars (March 12^th, Barga - Italy).
• 2015 Speaker - Seminar Series, Institute for Research in Immunology and Cancer, University of Montreal (April 24^th, Montreal - Canada).
• 2015 Speaker - MELA-BETES Joint Translational Call Meeting (March 20^th, Paris - France).
• 2014 Speaker - MELA-BETES Joint Translational Call Meeting (June 23^rd, Munich - Germany).
• 2013 Speaker - MELA-BETES Joint Translational Call Meeting (October 3^rd, Montreal - Canada).
• 2012 Speaker - MELA-BETES Joint Translational Call Meeting (December 21^st, Dresden - Germany).
• 2005 Selected Short Talk - 7^eJournée Scientifique de l’Équipe de Physiopathologie Endocrinienne (May, Lac Brome - Canada).
• 2004 Selected Short Talk - 6^eJournée Scientifique de l’Équipe de Physiopathologie Endocrinienne (September, Lac Brome - Canada).