An integrative interactomic approach to assess the function of CASK and its involvement in neurodevelopmental disorders

Proteins act in intertwined regulated networks and are the main effectors of biological processes, as well as the principal targets of drug treatments. Identification all the protein-protein interactions (PPIs) established by a protein in a particular context, its interactome, is a powerful approach to uncover its normal and pathological functions. We implemented a multipronged interactomic approach to decipher the functions of CASK (Ca2+/calmodulin-dependent serine protein kinase) and its involvement in some neurodevelopmental disorders (NDDs). CASK is a large multi-domain protein achieving multiple functions in the central nervous system (Figure 1A). Several severe NDDs are caused by CASK mutations whose dysfunctional consequences remain sketchy characterized, yet underpinning PPI perturbations are suspected.

We generated a contextual and quantitative interactome of the CASK wt protein of unprecedent depth and robustness, by combining four complementary interactomic approaches: affinity-purification mass spectrometry (AP-MS), in vitro AP-MS (pouring cell lysates on immobilized recombinant bait column), proximity-dependent biotinylation (BioID), and our recently developed Holdup assay (a robust and precise chromatographic assay for high-throughput quantification of protein-ligand affinities, Kd). Our CASK interactome is highly relevant since significantly enriched in (i) previously identified CASK partners; (ii) GO terms associated to the known functions of the protein, and (iii) NDD- or ASD- (Autism Spectrum Disorders) associated genes (Figure 1B). Strikingly, this interactome also reveals a potential new, previously undescribed function of the protein, which calls for further investigation. Finally, we systematically and finely investigated the complementarity between the four interactomic methods we used, providing a rigorous analysis of the strengths and weaknesses inherent to each approach (Figure 1C).

In the second step, we aim to demonstrate that some CASK-associated mutations exert their pathological mechanisms through global interactome perturbations (Figure 1D). We will analyze how these interactome perturbations correlate to phenotypic alterations. In the future, this pathological fingerprint of interactome perturbations will allow us to assess the pathogenicity of Variants of Unknown Significance (VUS), a critical question for diagnosing NDD. Together, our results will provide an in-depth understanding of CASK mutation-associated mechanisms.