S-Nitrosylation of p53 in Melanoma Cells Under Nitrosative Stress

Nitric oxide (NO) modifies protein structure and plays a critical role in promoting tumor growth, metastasis, and resistance to therapy. In inflammatory tumors, multiple key proteins, including p53, are susceptible to S-nitrosylation (SNO) by NO. In this study, we investigated the effects of different NO donors on melanoma cell growth and p53 modification. Biotin switch assays demonstrated that treatment with S-nitrosoglutathione (GSNO) or diethylenetriamine (DETA) NONOate significantly increased total S-nitrosylated proteins in A375 and SB2 melanoma cells. p53 was confirmed to undergo SNO under nitrosative stress, as evidenced by biotin switch assays and mass spectrometry. Proteomic analysis identified Cys242, Cys275, and Cys277 as specific SNO sites on p53. Structural modelling revealed that SNO of Cys242 and Cys277 disrupts p53 conformation and impairs its DNA-binding ability. These findings uncover a critical mechanism by which nitrosative stress alters the function of p53 through site-specific SNO, laying a foundation for further exploration of how SNO affects p53-mediated transcriptional regulation in melanoma cells.