Background: Ubiquitin-specific protease 9X (USP9X) is a deubiquitinating enzyme (DUB). USP9X haploinsufficiency causes USP9X Syndrome, a female neurodevelopmental disorder (NDD). Given that USP9X has >100 protein interactors involved in diverse biological processes, its deficiency likely impacts the proteome, thereby disrupting signal transduction as well as the transcriptome and epigenome.
Aim: Investigate the involvement of USP9X in NDDs using multi-omics.
Methods: Fibroblasts derived from four individuals with USP9X Syndrome were subjected to proteomic, transcriptomic and epigenomic profiling. APEX2-based proximity ligation assays were employed to identify USP9X interactors and interrogate the function of the USP9X ubiquitin-like (UBL) domain and phosphorylation events.
Results: Preliminary analysis of the proteomes of USP9X Syndrome fibroblasts revealed differential abundance of 1,354 proteins compared to controls. Of these, 253 were decreased and formed a highly connected protein-protein interaction network involved in cytoskeletal pathways. Further investigation revealed that USP9X is phosphorylated upon TGF-β stimulation in HEK cells. An APEX2-based USP9X biotin ligation assay was optimised, with pilot experiments confirming the ability of APEX2-USP9X to isolate known substrates, including SMURF1 and CEP55, in HEK cells. 126 biotinylated proteins, identified by MS with high confidence, were enriched in the pathways related to protein folding and ubiquitination.
Conclusions: Preliminary proteomics data suggest that USP9X haploinsufficiency leads to widespread changes in the proteome, which will be further compared with transcriptomic and epigenomic signatures. The biotin ligation technology has proven effective in labelling USP9X substrates, which opens opportunities to identify the function of USP9X phosphorylation and UBL domain on its protein-protein interactions.