Integrated quantitative proteomics and phosphoproteomics analysis reveals USP46-POU4F1-HPSE signaling axis in the...

Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of enteric ganglion cells in the distal colon, resulting in functional intestinal obstruction. While genetic mutations and microenvironmental imbalances have been implicated in HSCR, the underlying molecular mechanisms are not fully understood. This study uses integrated quantitative proteomics and phosphoproteomics analyses to characterize the differential protein profiles and phosphorylation modifications associated with HSCR. These findings reveal significant dysregulation of the extracellular matrix (ECM) remodelling pathway, suggesting its potential involvement in HSCR pathogenesis. Notably, the deubiquitinating enzyme USP46 is found to be significantly reduced in the aganglionic segments of HSCR patients. Through IP-MS, GST pull-down, and co-immunoprecipitation assays, it is demonstrated that USP46 interacts with the transcription factor POU4F1. Mechanistically, USP46 stabilizes POU4F1 via deubiquitination, increasing its binding to the heparanase (HPSE) promoter and increasing HPSE expression, which in turn promotes ECM remodelling and neural cell migration. The role of the USP46-POU4F1-HPSE signaling axis in HSCR pathogenesis is confirmed via chromatin immunoprecipitation-qPCR, luciferase reporter assays, and transwell migration assays. This study elucidates a novel regulatory mechanism linking USP46-mediated protein stabilization to ECM dynamics and neural cell migration, offering new insights into HSCR pathogenesis and potential therapeutic targets.