Identification of Protein Substrates Downstream of the deubiquitylase USP27X — 81p — Cheyenne Smith, Intisar Koch, Venkateshwarlu Bandi, Oduduabasi Isaiah

X-linked intellectual developmental disorder 105 (XLID105) is a recently described X-linked rare disease. Affected children display intellectual disability impairing both speech and behavior, and global developmental delay. This disorder is caused by variants in the USP27X gene, however the cellular and molecular mechanisms altered by these variants are currently unknown and are key to understanding XLID105 pathogenesis. Because USP27X is a deubiquitinating enzyme that catalyze ubiquitin removal and stabilization of protein substrates, we hypothesize that disruption of USP27X dysregulates its substrates and ubiquitin-related signaling pathways relevant for neurodevelopment. 

In this research, we used tandem ubiquitin binding entities (TUBE)-based global ubiquitin proteomics to unbiasedly identify putative USP27X substrates in mouse embryonic stem cells (ESC). We aim to validate the identified protein candidates as bona fide USP27X substrates by i) cloning the candidates in expression vectors, and ii) determining the effect of USP27X function in their ubiquitylation. Here we showed that we successfully cloned 11 candidate substrates in our PCAGGS Myc vector. Our preliminary results with TUBE pulldown assays indicate that the phosphopantetheinyl transferase Aasdhppt ubiquitylation is dependent on USP27X. USP27X substrates identified here will open new research directions in our lab and will be candidates to be exploited as therapeutic targets in XLID105. 

Sanford Research
Dr. Francisco Bustos