A ZDHHC3-USP5-PTRF axis links palmitoylation to ferroptosis-associated phenotypes in melanoma.

Melanoma is a highly aggressive malignancy with poor prognosis and therapy resistance. Although deubiquitinating enzymes (DUBs) regulate protein stability and cancer progression, their modulation in melanoma remains unclear. Here, we characterized ubiquitin‑specific peptidase 5 (USP5) using proteomic, biochemical, and functional approaches. USP5 was upregulated in melanoma and promoted proliferation, migration, and invasion. Mechanistically, USP5 directly interacted with and deubiquitinated PTRF, preferentially removing K48‑linked polyubiquitin chains to reduce proteasomal degradation. PTRF represents a functionally relevant substrate, though additional targets may exist. USP5 stability itself depended on S‑palmitoylation mediated by the palmitoyltransferase ZDHHC3, which suppressed USP5 ubiquitination and turnover. Silencing ZDHHC3 destabilized USP5, reduced PTRF levels, and impaired tumor growth. Perturbing the ZDHHC3-USP5-PTRF axis modulated ferroptosis‑associated phenotypes in vitro and affected xenograft tumor growth, accompanied by altered oxidative damage markers indicative of redox and lipid peroxidation changes, without directly establishing ferroptosis dependency. Our findings identify ZDHHC3‑mediated palmitoylation as a key determinant of USP5 stability and reveal a palmitoylation-deubiquitination cascade with PTRF as a downstream effector, linking this pathway to ferroptosis‑related cellular phenotypes. Further in vivo studies are needed to clarify mechanistic specificity and ferroptosis dependency.