ED Sciences Chimiques
Chemical biology approaches to quantify protein S-acylation
by Chloé FREYERMUTH--REYMOND (Institut de Chimie & de Biologie des Membranes & des Nano-objets)
The defense will take place at 9h00 - Amphithéâtre IECB, 2 Rue Robert Escarpit 33607 PESSAC FRANCE
in front of the jury composed of
- Emmanuelle THINON - Chargée de recherche - Chimie et Biologie des Membranes et Nanoobjets (CBMN) - Directeur de these
- Laurence ABRAMI - Maîtresse de recherche - Ecole polytechnique fédérale de Lausanne (EPFL) - Rapporteur
- Edward TATE - Professor - Imperial College London - Rapporteur
- Agnès RöTIG - Directrice de recherche - Institut Imagine - Examinateur
- Frédéric FRISCOURT - Associate Professor - Institut des Sciences Moléculaires (ISM) - Examinateur
- Gilles GUICHARD - Directeur de recherche - Chimie et Biologie des Membranes et Nanoobjets (CBMN) - Examinateur
S-acylation is a post-translational modification of proteins involving the covalent attachment of a fatty acid to cysteine residues. This addition of a hydrophobic moiety can alter the protein localisation, and can also affect their structure and/or stability. The reversibility and dynamism of this enzymatic modification enable it to play a regulatory role in cellular processes, with involvement in various biological functions. Aberrant S-acylation has been linked to a variety of human diseases, including cancers or neurodegenerative diseases. Existing proteomics methods are mostly based on relative quantification of the S-acylation of proteins. Tools to precisely quantify changes in S-acylation levels of each cysteine residue are noticeably lacking. This thesis project focuses on the development of a method to quantify the S-acylation levels of cysteine residues in a single proteome sample. The method relies on the sequential and differential labelling of free cysteines and S-acylated cysteines using a pair of isotopically-tagged chemical probes. The probes have identical structures, each composed of a cysteine-reactive electrophile and an alkyne handle, with a mass difference introduced by light and heavy isotopes (“light” probe (12C, 14N) and “heavy” probe (13C, 15N)). The labelled cysteines are then coupled by click reaction to an azide-capture reagent bearing a biotin moiety. Following tryptic digestion, cysteine-containing peptides are enriched using NeutrAvidin beads, and the eluted labelled peptides are analysed by LC-MS/MS. MS analysis provides heavy-to-light ratios for each labelled cysteine within a single proteome sample, which reveals the percentage of S-acylation of the cysteines. The chemical tools were synthesised and the workflow was developed with the selection of protein treatments' parameters using qualitative analysis approaches (e.g., Western blotting). Proteomics analyses allowed further refinement of key variables, including the combination of probes and capture reagents, the LC-MS/MS analysis parameters, and the downstream data processing to calculate the S-acylation percentages. The method was successfully applied to detect changes in S-acylation levels upon an external stimulus, providing new insights into associated signalling pathways and biological processes. Optimisations of the workflow will be pursued, notably by comparing new probes and automating the pipeline. We anticipate that the developed method will have broad applications for studying the S-acylation levels of cysteine residues and their role in different pathologies, potentially revealing innovative treatments.
ED Sciences de la Vie et de la Santé
ACTION OF 5-HT2A RECEPTORS ON NEUROTRANSMITTER SYSTEMS IN THE MOUSE BRAIN: APPLICATION TO PSYCHEDELICS.
by Jasmine BUTLER (Institut de neurosciences cognitives et intégratives d'Aquitaine)
The defense will take place at 14h00 - Amphithéatre (BBS - Salles RDC) Laboratoire INCIA - CNRS - UMR 5287 Bâtiment BORDEAUX BIOLOGIE SANTE 2 rue Docteur Hoffman Martinot BP 22 33076 Bordeaux cedex
in front of the jury composed of
- Philippe DE DEURWAERDERE - Full professor - Université de Bordeaux - Directeur de these
- Muriel DARNAUDERY - Full professor - Université de Bordeaux - Examinateur
- Carine BÉCAMEL - Docteure - Université de Montpellier - Rapporteur
- Mickael NAASSILA - Full professor - Université de Picardie Jules Verne (UPJV) - Rapporteur
- Nasser HADDJERI - Directeur de recherche - Université Claude Bernard Lyon 1 - Examinateur
The serotonin 2A receptor subtype (5-HT2AR) has gained interest following a resurgence of clinical and pre-clinical research on serotonergic psychedelics, compounds with converging agonist action on the 5-HT2AR. Psychedelics have antidepressant and anxiolytic properties, particularly when paired with therapy. Antagonism at the 5-HT2AR, as part of the pharmacological profile of atypical antipsychotics, may have benefits for schizophrenia and bipolar disorder. Despite their clinical relevance, the known impact of 5-HT2ARs on brain function, particularly neurotransmission, is limited. Functional brain networks have been conceptualised by correlating electrical or metabolic neuroimaging signals between brain regions using functional magnetic resonance imaging or encephalography. These studies have shown that psychedelic 5-HT2AR agonists alter the connectivity of these brain-wide networks. However, neuroimaging is currently unable to assess the neurochemistry of neurotransmitter systems and their interplay. This thesis addresses the hypothesis that psychedelic 5-HT2AR agonists disrupt the functional organization of brain-wide neurotransmitter systems. Tissue content of both classical (glutamate and GABA) and monoaminergic (serotonin, dopamine and noradrenaline) neurotransmitters and their metabolites were measured in 28 regions of the mouse brain following high-affinity 5-HT2AR agonist and antagonist, TCB-2 and MDL100,907 respectively. To promote a coherent organisation of neurotransmitter systems mice were placed in a forced exploration paradigm and their behaviour was filmed before post-mortem neurochemical quantification. A significant challenge of this thesis lies in manipulating such a large neurochemical dataset that, beyond quantitative modulation, allows for a correlative approach incorporating graph theory to build networks of neurochemical connectivity. This novel analysis prompted the development of code to accompany the launch of a neurochemical database, including this dataset, making analysis using this new approach accessible. The obtained results demonstrate that a variety of compounds across the 28 brain regions form distinct neurobiological networks that can be monitored with high-pressure liquid chromatography coupled to electrochemical detection. A striking density of neurochemical correlations between brain regions in vehicle-treated animals was observed, with a distinct regional organisation for dopamine and noradrenaline. The 5-HT2AR agonist TCB-2 (0.3, 3, and 10 mg/kg) as well as the 5-HT2AR antagonist MDL-100,907 (0.2 mg/kg) reduced the number of correlations and disrupted the organisation of correlations for all neurotransmitters across the brain. Some effects of TCB-2, notably on serotonergic parameters were independent of 5-HT2ARs in several brain regions. Other effects including behavioural parameters such as head twitches or components of the exploratory behaviour, as well as the levels of serotonin, dopamine, and noradrenaline in the anterior cingulate cortex were reduced by MDL-100,907 pretreatment. MDL-100,907 alone had very few effects on the quantity of neurochemicals across brain regions. Overall, this thesis highlights that 5-HT2ARs likely play an important role in organising the coherence of neurotransmitter systems in response to a forced exploratory behaviour whether or not it is associated with quantitative changes. The thesis offers a new paradigm to address the function of neurotransmitter systems. It enlarges the understanding of the mechanism of psychedelic action in the brain including vast brain territories (sensory, motor, cognitive) with some lateralized effects, and altered connectivity of neurotransmission systems.