Phosphoproteomic profiling highlights CDC42 and CDK2 as key players in the regulation of the TGF-β pathway inALMS1andBBS1knockout models

BACKGROUNDThe primary cilium is a sensory organelle that extends from the plasma membrane. It plays a vital role in physiological and developmental processes by controlling different signalling pathways such as WNT, Sonic hedgehog (SHh), and transforming growth factor β (TGF-β). Ciliary dysfunction has been related to different pathologies such as Alström (ALMS) or Bardet-Biedl (BBS) syndrome. The leading cause of death in adults with these syndromes is chronic kidney disease (CKD), which is characterised by fibrotic and inflammatory processes often involving the TGF-β pathway.METHODSUsing genomic editing with CRISPR-CAS9 and phosphoproteomics we have studied the TGF-β signalling pathway in knockout (KO) models forALMS1andBBS1genes. We have developed a network diffusion-based analysis pipeline to expand the data initially obtained and to be able to determinate which processes were deregulated in TGF-β pathway. Finally, we have analysed protein-protein interactions to prioritise candidate genes in the regulation of the TGF-β pathway in Alström and Bardet-Biedl syndrome.RESULTSAnalysis of differentially phosphorylated proteins identified 10 candidate proteins in theALMS1KO model and 41 in theBBS1KO model. After network expansion using a random walk with a restart model, we were able to obtain processes related to TGF-β signalling such as endocytosis in the case ofALMS1or extracellular matrix regulation inBBS1. Protein interaction analyses demonstrated the involvement of CDC42 as a central protein in the interactome inALMS1and CDK2 in the case ofBBS1.CONCLUSIONIn conclusion, the depletion ofALMS1andBBS1affects the TGF-β signalling pathway, conditioning the phosphorylation and activation of several proteins, including CDC42 in the case ofALMS1and CDK2 in the case ofBBS1.