Ubiquitin-proteasome-dependent IMPDH2 degradation regulated by ANKRD9 controls myogenic differentiation in Gallus...

The ubiquitin-proteasome system (UPS) is a master regulator of protein homeostasis during skeletal muscle development, yet the upstream molecular sensors that couple UPS activity to the myogenic program remain incompletely understood. Here, we identify ANKRD9 (Ankyrin Repeat Domain 9) as a novel pro-myogenic regulator in Gallus gallus that orchestrates myoblast differentiation by targeting inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) for ubiquitin-mediated proteasomal degradation. We demonstrate that ANKRD9 mRNA and protein levels are dynamically upregulated during chicken myogenic differentiation, reaching a 3.8-fold peak at 72 hours post-induction. Gain- and loss-of-function experiments establish that ANKRD9 overexpression promotes myotube formation (fusion index +41.3%; p < 0.01), while ANKRD9 knockdown impairs differentiation (-38.7%; p < 0.01). Co-immunoprecipitation coupled to LC-MS/MS proteomics and proximity ligation assay reveal a direct physical interaction between ANKRD9 and IMPDH2, a rate-limiting enzyme of the guanine nucleotide synthesis pathway. ANKRD9 overexpression reduces IMPDH2 protein half-life from 8.4 h to 3.1 h in a proteasome-dependent, lysosome-independent manner. In vivo ubiquitination assays and mass spectrometric diglycine remnant profiling identify lysine K414 as the primary ubiquitination site on IMPDH2. Critically, the non-ubiquitinable IMPDH2-K414R mutant is resistant to ANKRD9-induced degradation and fully rescues the enhanced differentiation phenotype of ANKRD9-overexpressing myoblasts, establishing a linear ANKRD9→UPS→IMPDH2 epistatic axis. IMPDH2 itself functions as a negative regulator of myogenesis through GTP-dependent mechanisms, and its pharmacological inhibition by mycophenolic acid phenocopies pro-differentiation effects. Pan-transcriptomic RNA-seq analysis confirms coordinated upregulation of UPS components, E3 ubiquitin ligases MuRF1 and MAFbx, and classical myogenic regulatory factors by ANKRD9. Collectively, our findings unveil a previously uncharacterized ANKRD9-IMPDH2-UPS axis that integrates purine metabolism with proteasomal proteolysis to control avian skeletal muscle differentiation, with implications for poultry muscle biology and vertebrate myogenesis.