Screening for Protein-Protein Interaction Inhibitors Using a Bioluminescence Resonance Energy Transfer (BRET)–Based Assay in Yeast

Corbel, Caroline; Sartini, Sara; Levati, Elisabetta; Colas, Pierre; Maillet, Laurent; Couturier, Cyril; Montanini, Barbara; Bach, Stéphane

doi:10.1177/2472555216689530

Cited by 15 publications

(11 citation statements)

References 15 publications

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“…An example of these interactions is that established by the enzyme adenosine deaminase with different proteins ( Cortés et al, 2015 ). Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are biophysical techniques widely used to analyze direct protein–protein interactions that take place in living cells as well as conformational changes within proteins or molecular complexes ( Ciruela, 2008 ; Kimura et al, 2010 ; Kim et al, 2011 ; Lohse et al, 2012 ; Deriziotis et al, 2014 ; Brown et al, 2015 ; Mo and Fu, 2016 ; Corbel et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

Moreno¹,

Canet²,

Gracia³

et al. 2018

Front. Pharmacol.

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Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

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Section: Introductionmentioning

confidence: 99%

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

Moreno¹,

Canet²,

Gracia³

et al. 2018

Front. Pharmacol.

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“…L'identification puis le clonage des acteurs moléculaires à l'origine de cette luminescence (tels que mérisation du RCPG de la phéromone α [56]. En tenant compte des avantages qu'offre le champignon, le BRET a ensuite été utilisé pour le criblage d'inhibiteurs d'IPP [57]. C'est principalement l'utilisation d'une levure mutante erg6 (Delta(24)-sterol C-methyltransferase), rendue hyper-perméable par altération de sa membrane due à un défaut d'ergostérol, et le criblage effectué sur des complexes protéiques non préformés (la molécule à tester est mise en contact avec la chimère protéique « acceptrice » avant l'induction de l'expression de la chimère « donneuse ») qui ont contribué au succès de cette approche.…”

Section: La Levure : Un Outil Pour La Recherche Sur Les Interactions unclassified

“…Plusieurs criblages ont été mis en oeuvre afin d'identifier des inhibiteurs de l'interaction CDK5/p25, une protéine kinase impliquée notamment dans divers troubles neurodégénératifs [58,59] et de nouveaux antibactériens, inhibiteurs de l'interaction entre la sous-unité b' et le facteur d'initiation s 70 de l'ARN polymérase bactérienne [60]. L'utilisation de nouveaux substrats de luciférase, de la NanoLuc® et de nouveaux couples donneur/ accepteur (mutants de la luciférase et de l'accepteur, tels que la mVenus ou encore la mTurquoise) permettront d'augmenter la sensibilité et l'usage du BRET dans la levure pour y étudier de nouvelles IPP [57].…”

Section: La Levure : Un Outil Pour La Recherche Sur Les Interactions unclassified

La levure modèle et outil… aussi pour la recherche thérapeutique

2020

Self Cite

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La levure a été utilisée de façon empirique pendant des millénaires pour la panification et la fermentation des sucres en alcool. C’est seulement à partir de 1857 que Louis Pasteur décrit le microorganisme à l’origine de ces deux activités agroalimentaires majeures. Dès lors, les souches de levure ont pu être sélectionnées et modifiées sur une base rationnelle pour optimiser leurs usages agroalimentaires, permettant ainsi l’essor de la levure comme modèle biologique eucaryote. Cette utilisation a conduit à de très nombreuses découvertes de biologie cellulaire fondamentale. Depuis une vingtaine d’années, la levure est également utilisée comme modèle et outil pour la santé humaine. Ces approches s’étendent de la production de molécules thérapeutiques à la recherche de candidats-médicaments et de sondes chimiques, en passant par la mise au point de tests diagnostiques et la découverte de nouvelles cibles thérapeutiques. Cette utilisation de la levure en chémobiologie fait l’objet de la présente revue.

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“…If the RLuc8 donor fusion molecule catalyzed the coelenterazine 400a and the GFP 2 acceptor fusion molecule is located less than 10 nm from the donor, a transfer of energy will occur from the donor to the acceptor. This method has been widely used to study PPI (Bery et al., ; Felce et al., ; Mercier et al., ) and also PPI inhibition by small molecules (Beautrait et al., ; Corbel et al., ; Corbel et al., ; Lavoie et al., ; Mazars & Fahraeus, ; Quevedo et al., ) or macromolecules (Bery et al., ; Guillard et al., ; Spencer‐Smith et al., ). It offers several advantages: it is a very sensitive technique as it is based on luminescence, there is no background signal from cellular autofluorescence.…”

Section: Commentarymentioning

confidence: 99%

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

Béry

Rabbitts

2019

CP Cell Biology

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Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. C 2019 by John Wiley & Sons, Inc.Keywords: biosensors r BRET r protein-protein interaction inhibition r RAS family GTPases of 22RAS associating (RA) domain of RALGDS. We also used as acceptor the full-length CRAF and PI3Kα effectors to study their respective downstream signaling pathways. Therefore, using this BRET-based RAS biosensors toolbox, we characterized the cellular properties of various RAS inhibitors comprising anti-RAS Design Ankyrin Repeat Proteins (DARPins) (Guillard et al., 2017) and anti-RAS intracellular domain antibody (iDAb RAS) (Bery et al., 2018) macromolecules and antibody derived anti-RAS compounds Quevedo et al., 2018).Here we describe a set of protocols that include step-by-step instructions to monitor the interaction between RAS and a partner (Basic Protocol 1), to identify inhibitors of RAS PPIs including macromolecules (Basic Protocol 2) and small molecules (Basic Protocol 3). We also describe a protocol to detect the effect of RAS inhibitors on the RAS downstream pathways by immunoblot (Basic Protocol 4) and finally a protocol explaining how to calculate the BRET ratio obtained in the previous protocols (Basic Protocol 5). Figure 1Use of the BRET-based RAS biosensors to assess a PPI. (A) A schematic representation of the BRET-based biosensors. A protein (P1) is fused to the donor moiety RLuc8 and a protein (P2 or P3) is tagged with the acceptor moiety GFP 2 . If P1 does not bind to P2, it only produces a background BRET signal. However, when P1 interacts with P2, it induces a BRET signal, if the RLuc8 and GFP 2 domains are within 10 nm. This BRET signal can be decreased by addition of a competitor (either by a macromolecule or a small molecule inhibitor). (B) A representative donor saturation assay between KRAS G12D , KRAS WT and KRAS S17N (donors) and CRAF RBD (acceptor) with total GFP 2 and RLuc8 controls. Where error bars are presented, these correspond to mean values ± SD of quadruplicates technical repeats. Day 2: Cell transfection2. Mix together a fixed amount of RLuc8 construct (typically 50 ng) and a varying amount of GFP 2 construct (see quantity in Table 2).The pEF-myc-cyto empty plasmid is used to equalize total amount of transfected DNA between wells. The total DNA amount transfected per well is 1.6 μg. ...

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Screening for Protein-Protein Interaction Inhibitors Using a Bioluminescence Resonance Energy Transfer (BRET)–Based Assay in Yeast

Cited by 15 publications

References 15 publications

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

La levure modèle et outil… aussi pour la recherche thérapeutique

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

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