Endogenous peptide derived from c-Cbl-associated protein counteracts its inhibitory effect on enteric neural crest cell colonization in Hirschsprung disease

Hirschsprung disease (HSCR) is a congenital malformation characterized by the absence of the enteric nervous system (ENS) in the distal colon, resulting from defective colonization of enteric neural crest cells (ENCCs). The underlying pathogenesis of HSCR remains incompletely understood. Here, we report that c-Cbl-associated protein (CAP), also known as sorbin and SH3 domain-containing protein 1 (SORBS1), is upregulated in the aganglionic colon tissues of children with HSCR. Functional studies revealed that CAP overexpression suppresses ENCC colonization by binding the lipid raft protein flotillin-1 through its sorbin homology (SoHo) domain, followed by recruitment of the focal adhesion protein vinculin via its SH3 domain. Using mass spectrometry, we identified an endogenous CAP-derived peptide, termed PDCAP, in aganglionic colon tissues. ELISA further revealed reduced PDCAP levels in the diseased colon tissues of HSCR children. Mechanistically, PDCAP exerts a protective role by competing with its precursor protein, CAP, for binding to flotillin-1, thereby reversing CAP-mediated inhibition of ENCC colonization. This protective function was further validated in Cap lsl/lsl ;Nestin-Cre as well as Ednrb -/- mouse models of either sex, where PDCAP promoted ENCC colonization and ENS development. Collectively, our findings establish PDCAP as a functional antagonist of its precursor CAP, providing a rationale for exploring peptide-mediated interventions in HSCR. Significance Statement This study identifies a novel pathogenic mechanism in Hirschsprung disease (HSCR), showing that upregulated c-Cbl-associated protein (CAP) suppresses enteric neural crest cell (ENCC) colonization through interactions with flotillin-1 and vinculin. We further characterize an endogenous peptide derived from c-Cbl-associated protein (PDCAP) that competitively binds flotillin-1 and thereby counteracts CAP-mediated inhibition of ENCC colonization. By demonstrating the protective role of PDCAP in promoting ENCC colonization and enteric nervous system (ENS) development, our findings suggest a potential biological strategy to support ENS formation and provide a new direction for future therapeutic exploration.