Caractérisation de l’activité Nur77 et de ses complexes en essais BRET par complémentation
In the central nervous system, dopamine plays a critical role in many physiological functions such as learning, voluntary movement, motivation, cognition and hormone production. It was also shown that the dopamine signaling system is altered in many neurological and psychiatric disorders like Parkinson's disease and schizophrenia. Studies conducted in the laboratory of Dr. Daniel Lévesque (host laboratory) have shown that nuclear receptors Nur77 (NR4A1, NGFI-B) and RXRγ (retinoid X receptor γ isoform) are involved in the regulation of dopamine effects. These data suggest that the Nur77/RXR complex plays a crucial role in the effect of antipsychotic and anti-parkinsonian drugs. However, very few drugs targeting Nur77 have been identified to date and drugs acting at RXRγ remain poorly characterized. Furthermore, currently available tests cannot recapitulate the complexity of nuclear receptor activities and generate indirect measures of drug activities. To better understand Nur77/RXRγ complex activity we developed a new and original Bioluminescence Resonance Energy Transfer (BRET)-luciferase Protein Complementation Assay (PCA) based on the recruitment of a co-activator motif fused with YFP. The assays have been validated by dose-response curves using different RXR compounds. EC50 obtained were very similar to the values previously reported in the literature. We were also able to identify a compound, SR11237 (BMS649), which appears to have specificity for the complex Nur77/RXRγ compared to Nurr1/RXRγ and RXRγ/RXRγ. Our results indicate that these BRET assays can be used to evaluate the selectivity of compounds at Nur77/RXRγ, Nurr1/RXRγ or RXRγ/RXRγ complexes.  Another aspect of my PhD project was to better understand how Nur77 activity is regulated by post-translational modifications. We were able to highlight the importance of Nur77 SUMOylation in the activity of monomeric, homodimer and heterodimer forms of Nur77 using various BRET assays. We identified that Nur77 preferentially recruits SUMO2 mainly on its lysine 577 residue. It is interesting to note that SUMO2 recruitment by Nur77 is potentiated in the presence of SUMO E3 ligase PIAS4 (PIASγ). Also, the loss of SUMOylation on lysine 577 strongly reduced co-activator motif recruitment by monomeric form of Nur77, but not for homo- and hetero-dimer complexes. However, PIAS4 by itself does not potentiate co-activator recruitment, suggesting that this SUMO E3 ligase is only involved in the recruitment process of SUMO, but not in the co-activator recruitment. Thus, we identified a new post-translational modification on Nur77 that specifically regulates its monomeric activity.  These results provide critical new data that will help to identify new compounds targeting selective nuclear receptor complexes (Nur77/RXR) that may improve the treatment of Parkinson's disease and schizophrenia, as well as to get a better understanding of the mechanisms regulating the activity of Nur77.
Nur77 RXRγ Parkinson Dopamine SR11237 BRET SUMOylation Schizophrenia