Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein–Protein Interactions in Human Living Cells

International audienceAbstract Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia ( Antp ) and Ultrabithorax ( Ubx ) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster . We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx . We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution

The human HOXA9 protein uses paralog-specific residues of the homeodomain to interact with TALE-class cofactors

Abstract HOX proteins interact with PBX and MEIS cofactors, which belong to the TALE-class of homeodomain (HD)-containing transcription factors. Although the formation of HOX-PBX complexes depends on a unique conserved HOX motif called hexapeptide (HX), the additional presence of MEIS induces a remodeling of the interaction, leading to a global dispensability of the HX motif for trimeric complex formation in the large majority of HOX proteins. In addition, it was shown that the anterior HOXB3 and central HOXA7 and HOXC8 proteins could use different alternative TALE interaction motifs, with or without the HX motif, depending on the DNA-binding site and cell context. Here we dissected the molecular interaction properties of the human posterior HOXA9 protein with its TALE cofactors, PBX1 and MEIS1. Analysis was performed on different DNA-binding sites in vitro and by doing Bimolecular Fluorescence Complementation (BiFC) in different cell lines. Notably, we observed that the HOXA9-TALE interaction relies consistently on the redundant activity of the HX motif and two paralog-specific residues of the HOXA9 HD. Together with previous work, our results show that HOX proteins interact with their generic TALE cofactors through various modalities, ranging from unique and context-independent to versatile and context-dependent TALE binding interfaces

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors

Summary: HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors. : Dard et al. examine interaction flexibility between HOX proteins and TALE cofactors. The authors identify specific TALE-binding sites in different human HOX proteins. Most of these TALE-binding sites correspond to short motifs that are differently used depending on the DNA-binding site or cell context. Keywords: HOX, PBX, MEIS, BiFC, SLiM, TALE, homeodomain, transcription, protein interactio

Role of a versatile peptide motif controlling Hox nuclear export and autophagy in the Drosophila fat body

International audienceHox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deletion forms of the Drosophila Hox protein Ultrabithorax (Ubx), we identified the presence of an unconventional nuclear export signal (NES) that overlaps with a highly conserved motif originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors that act in development and cancer. We show that this unconventional NES is involved in the interaction with the major exportin protein CRM1 (also known as Embargoed in flies) in vivo and in vitro We find that this interaction is tightly regulated in the Drosophila fat body to control the autophagy-repressive activity of Ubx during larval development. The role of the PBC interaction motif as part of an unconventional NES was also uncovered in other Drosophila and human Hox proteins, highlighting the evolutionary conservation of this novel function. Together, our results reveal the extreme molecular versatility of a unique short peptide motif for controlling the context-dependent activity of Hox proteins both at transcriptional and non-transcriptional levels

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

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 developing tools that allow capturing transient and low affinity protein-protein interactions (PPIs) in live conditions. Here we present an experimental strategy, Cell-PCA (Cell Protein Complementation Assay), which is based on BiFC (Bimolecular Fluorescence Complementation) and high throughput sequencing for ORFeome-wide analyses of different interactomes in the same live cell context. The specificity and sensitivity of Cell-PCA was established by using a wild type and a single amino-acid mutated HOXA9 protein, and the approach was subsequently applied for seven additional human HOX proteins. These proofof-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 for promoting its proliferative activity in a cancer cell context. Taken together, our work demonstrates that Cell-PCA is pertinent for revealing and, importantly, comparing interactomes between 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

LACC1 deficiency links juvenile arthritis with autophagy and metabolism in macrophages

International audienceJuvenile idiopathic arthritis is the most common chronic rheumatic disease in children, and its etiology remains poorly understood. Here, we explored four families with early-onset arthritis carrying homozygous loss-of-expression mutations in LACC1. To understand the link between LACC1 and inflammation, we performed a functional study of LACC1 in human immune cells. We showed that LACC1 was primarily expressed in macrophages upon mTOR signaling. We found that LACC1 deficiency had no obvious impact on inflammasome activation, type I interferon response, or NF-κB regulation. Using bimolecular fluorescence complementation and biochemical assays, we showed that autophagy-inducing proteins, RACK1 and AMPK, interacted with LACC1. Autophagy blockade in macrophages was associated with LACC1 cleavage and degradation. Moreover, LACC1 deficiency reduced autophagy flux in primary macrophages. This was associated with a defect in the accumulation of lipid droplets and mitochondrial respiration, suggesting that LACC1-dependent autophagy fuels macrophage bioenergetics metabolism. Altogether, LACC1 deficiency defines a novel form of genetically inherited juvenile arthritis associated with impaired autophagy in macrophages