A nanobody based approach to capture interactomes of dimeric protein complexes in living cells

L’identité et le devenir de chaque cellule dépend du contenu en protéines et, en particulier, des réseaux d'interactions protéine-protéine (IPP, également appelés interactomes). Les protéines ont la propriété générale de s'engager dans des assemblages macromoléculaires très variés, chacun ayant des fonctions bien distinctes. Par conséquent, identifier les IPP et les lier à des complexes particuliers est un enjeu crucial mais difficile en biologie. Cette problématique a été au cœur de mon travail de doctorat. Une première partie de mon travail est dédiée à l'amélioration d'une méthode existante pour capturer de nouvelles IPP dans le contexte de fonctions biologiques définies. Ce travail a été réalisé avec ERK1, un régulateur clé en aval de plusieurs voies de signalisation impliquées dans de nombreux cancers. Les nouveaux outils ont été testés dans le contexte de fonctions de ERK1 sensibles à deux molécules inhibitrices dans les cellules humaines HEK293T. Une interaction a été confirmée aux niveaux fonctionnel et moléculaire, ainsi qu’en utilisant une stratégie d'imagerie originale pour accéder à la dynamique des IPP dans les cellules vivantes. La deuxième partie de mon travail de doctorat est dédiée à l'établissement d'une méthodologie pionnière pour capturer les IPP endogènes établies par un complexe protéique dimérique spécifique dans les cellules humaines vivantes. Cette méthodologie couple la Complémentation de Fluorescence Bimoléculaire (BiFC) et les technologies démarquage par la biotine de proximité. Plus précisément, elle repose sur l’utilisation d’un petit anticorps (appelé aussi « nanobody ») dirigé contre le complexe BiFC et fusionné à la ligase biotine TurboID. Ces outils ont été établis avec les complexes TAZ/14-3-3e et TAZ/TEAD2, qui traduisent respectivement l'activité de la voie de signalisation Hippo dans le cytoplasme et le noyau. Notre approche a permis de capturer les interactomes spécifiques de ces deux complexes protéiques et d'identifier un nouveau régulateur clé du complexe TAZ/14-3-3e pour contrôler ses fonctions de prolifération cellulaire. Dans son ensemble, mon travail de doctorat a introduit deux méthodologies complémentaires pour déchiffrer les réseaux d'IPP au niveau de fonctions biologiques spécifiques ou pour un complexe protéique spécifique en contexte cellulaire vivant. Ces approches offrent une nouvelle dimension pour comprendre les fonctions des protéines et les interactomes sous-jacents dans des contextes cellulaires normaux ou pathologiques.Cell fate and fitness depend on the protein content, and in particular on the interaction networks (also called interactomes) connecting the different proteins. Proteins have the general property to engage in diverse and occasionally overlapping macromolecular assemblies, each serving distinct purposes. Therefore, identifying protein-protein interactions (PPIs) and linking them to complexes is a crucial yet challenging issue in biology. This issue was at the core of my PhD work. The first part of my work was dedicated to the improvement of an existing method for capturing novel PPIs in the context of defined biological functions. This work was established with ERK1, which is a key downstream regulator of several signaling pathways involved in many different cancers. The new tools were tested in the context of two different inhibitory molecules to capture drug-sensitive interactions of ERK1 in human HEK293T cells. One such interaction was confirmed at the functional and molecular levels, by using an original imaging strategy to access the PPI dynamics in live cells. The second part of my PhD work was dedicated to the establishment of a pioneer methodology to capture endogenous PPIs established by a specific dimeric protein complex in human live cells. This methodology couples Bimolecular Fluorescence Complementation (BiFC) and proximity biotin labelling technologies. More specifically, it is based on a GFP-nanobody directed toward the BiFC complex and fused to the TurboID biotin ligase. Tools were established to map TAZ/14-3-3 and TAZ/TEAD complexes interactome, which translate the activity of the Hippo signaling pathway in the cytoplasm and nucleus, respectively. Our approach allowed capturing specific interactomes of the two dimeric protein complexes and identifying a novel key regulator of TAZ/14-3-3 complexes in a cancer cell context. Collectively, my PhD work introduced two complementary methodologies for deciphering PPI networks in the context of specific biological functions or in the context of a specific protein complex in human live cells. These approaches provide a novel dimension for understanding protein functions and the underlying interactomes in normal or pathological cell contexts

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein–protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context

Targeting ERK-MYD88 interaction leads to ERK dysregulation and immunogenic cancer cell death

International audienceThe quest for targeted therapies is critical in the battle against cancer. The RAS/MAP kinase pathway is frequently implicated in neoplasia, with ERK playing a crucial role as the most distal kinase in the RAS signaling cascade. Our previous research demonstrated that the interaction between ERK and MYD88, an adaptor protein in innate immunity, is crucial for RAS-dependent transformation and cancer cell survival. In this study, we examine the biological consequences of disrupting the ERK-MYD88 interaction through the ERK D-recruitment site (DRS), while preserving ERK’s kinase activity. Our results indicate that EI-52, a small-molecule benzimidazole targeting ERK-MYD88 interaction induces an HRI-mediated integrated stress response (ISR), resulting in immunogenic apoptosis specific to cancer cells. Additionally, EI-52 exhibits anti-tumor efficacy in patient-derived tumors and induces an anti-tumor T cell response in mice in vivo. These findings suggest that inhibiting the ERK-MYD88 interaction may be a promising therapeutic approach in cancer treatment