Development of high-throughput approaches for quantification of protein-ligand affinities
Development of high-throughput approaches for quantification of protein-ligand affinities
Describing cellular interactomes has been the goal of many high-throughput studies over the last decade, resulting in the identification of hundreds of thousands of binary interactions. Each of these techniques has its own strengths and flaws, resulting in a relatively low overlap between the published interactomes, and a still largely incomplete coverage of the human interactome. In addition, while affinities can span several orders of magnitude and are thus an essential parameter of the interactomes, interactomic data produced by these high-throughput experiments are almost exclusively qualitative ("binds" vs "does not bind"). Thus, the accurate description of protein interaction networks requires new approaches to address interactomes quantitatively, by measuring affinities at a proteome-wide scale.
To fill this gap our team has developed the Holdup, a robust and precise chromatographic assay for high-throughput quantification of protein-ligand affinities. Recently, the coupling of our method to a mass spectrometry readout has allowed to increase its multiplex and measure the interactions between a bait protein of interest and pools of thousands of potential preys in a single assay. Different types of preys have been successfully used: full-length proteins from total cell lysates for a discovery-driven approach, or pool of peptides / protein domains for quantifying interactions between interaction areas (fragmentomic approach). In close collaboration with teams specialized in mass-spectrometry, we are no pushing the limits of our method aiming for the best possible robustness, quantitative precision, proteome coverage, and throughput. We are also exploring the diversity of protein ligands for which the Holdup can quantify affinities: proteins but also DNA and RNA, as well as small molecules. Finally, we are also interested in assessing the complementarity of the Holdup assay with other interactomic methods, to provide a rigorous analysis of how these different methods complement one another as well as their potential bias.
Projets en cours
Quantitative interactomes, affinity variations and functional impacts (Shared project with Gilles Travé, Yves Nominé)
Living organisms are highly complex systems of interacting molecules, that go through highly reproducible cycles of self-organization and chemical reactions. Intermolecular binding energies (quantified by binding affinity constants, Kd) are the interaction potentials of these systems. Whenever intrinsic genomic sequences vary, some DNA, RNA and protein molecules vary in their extrinsic interaction potentials towards the rest of the system. According to the law of mass action, this should impact the relative concentrations of the various molecular complexes formed, and therefore the global organization and the overall reactions performed by the system should also vary. Ultimately, this should impact the behaviour of the system - in other terms, its biological functions, or phenotypic traits. As described in another paragraph of our team webpage, our lab is developing methods for measuring and quantifying all the binding energies of a given protein, RNA or DNA molecule towards the other molecules. In this way, we obtain "binding profiles" that quantify the intrinsic interaction potential of each protein/RNA/DNA molecule of the studied organism, towards all the other molecules in the system. Additionally, transcriptomic and proteomic approaches provide access to the abundances/concentrations of interacting molecules in particular cell populations (tissues, organs...) of the same organism.
In this project, we measure the binding profiles of large families of protein, RNA or DNA molecules, first using their "wild-type" sequences found most frequently in the human population. Then, we re-measure the same profiles for particular "variant" sequences found across the human population. As variant sequences may be categorized as benign, likely benign, pathogenic, likely pathogenic or of unknown significance, we generally pick various instances across these different categories. Once the profiles are measured, we first apply the law of mass action to predict how the variations of binding energies should affect the proportion of complexes in chosen instances well-characterized laboratory cell lines. Next, we establish cell lines where the wild-type sequences are replaced by the corresponding variants, and measure quantitative phenotypic traits of the variant cell lines, as compared to the wild-type cell lines. The quantitative phenotypic traits that we will systematically measure will be the proteomes and transcriptomes of the different cell lines. Finally, we seek potential correlations between the quantitative variations of binding energies of the variants, and the variations of quantitative traits expressed by the corresponding variant cell lines. The final aim is to investigate whether intrinsic, genetic-based variations of binding profiles can be used to predict extrinsic variations of phenotypes within complex cell populations.
Structural interactomics of particular neurodevelopmental disorders (shared project with Gilles Travé, Isabelle Billas)
Human brain development and functioning involve complex protein networks whose binding energies are finely tuned for optimal functioning. When, due to gene alterations, a key protein actor in such a network is absent, overexpressed or mutated, this may create a "network imbalance" and lead to a suboptimal brain development or functioning, which can cause neurodevelopment disease (NDD). Hence, we propose to shift the focus from "gene-to-disease" to "interactome-to-disease" relationship studies.
We are exploring this hypothesis by studies of Ube3A (~850 residues), HERC2 (~4800 residues) and CASK (~930 residues) proteins. Ube3A and HERC2 are both mutually-interacting E3 ubiquitin ligases belonging to the HECT domain family. Their coding genes are close neighbors in the 15q chromosomal region, and their genetic alteration (mutation, deletion or duplication) can lead to strong NDD such as Angelman, Angelman-like or Dup15q syndromes. In this project, we study by crystallography and cryo-EM the structures of Ube3A and HERC2, of their binding interface, and of their complexes with a variety of other human proteins. We explore the interactomes of wild-type or mutated Ube3A and HERC2, and quantify their cellular concentrations and binding affinities. We also perform cell localization and co-localization studies, and check whether best and most relevant binders are also involved in NDD, which appears to be often the case.
CASK is a highly conserved scaffold protein achieving multiple functions in and outside of the central nervous system, thanks to the variety of protein-protein interactions it establishes through its different domains. CASK is mutated in a range of severe NDD, such as X-linked intellectual disability with or without nystagmus (XLID), X-linked intellectual disability with Microcephaly and Pontine and Cerebellar Hypoplasia (MICPCH), amongst others. The wide variety of CASK-associated functions allows for multiple possible pathological mechanisms that remain elusive. Nonetheless, the loss of interaction of CASK with some of its well-known partners has been described for several disease-related mutants. In this project, we integrate different and complementary interactomic methods to generate a contextual and quantitative CASK interactome. We also quantify how CASK pathological variants affect this interactome, and how these interactome perturbations correlate with phenotypic alterations. We expect to define pathological fingerprint of interactome perturbations that will help in assessing the pathogenicity of variants of unknown significance (VUS), a critical question for diagnosing NDDs.