ChIP-Sequencing analysis of PPT1/LDHA binding DNA in HaCaT cells after 20 Gy iradiation

Radiation-induced skin injury is a common side effect of radiotherapy, which seriously affects the life quality of patients. Nitric oxide (NO) plays critical roles in redox signaling in normal and pathological cellular conditions and is also involved in protein S-nitrosylation modification, which regulates protein function via covalent reaction of NO-related species with a cysteine thiol group on the target protein. We have previously reported that ionizing radiation decreased NO levels by oxidation of BH4, a co-factor of eNOS. However, molecular change and underlying mechanisms of protein S-nitrosylation in the progression of radiation-induced skin injury is still an enigma. Here, we investigated the profiling of protein S-nitrosylation in irradiated skin fibroblast WS1 cells and a mouse model with radiogenic skin injury by iodoacetyl tandem mass tag (iodoTMT)-labeling protein quantification. It was found that ionizing radiation reduced the level of S-nitrosylation of a variety of proteins in the skin cells and tissues exposure to radiation. Among the dysregulated S-nitrosylated proteins, palmitoyl-protein thioesterase 1 (PPT1) was investigated in greater detail. Specifically, we found that ionizing radiation decreased the S-nitrosylation modification of PPT1. Ionizing radiation increased PPT1 expression and resulted in its nuclear translocation. To explore the mechanism underlying regulation of nuclear PPT1 functions, immunoprecipitation assay was performed with anti-PPT1, the precipitated proteins were subjected to western blot using an Coomassie blue staining to detect the PPT1-associated protein. Then the PPT1-associated proteins were analyzed by utilizing a tandem mass spectrometry (MS/MS). As expected, the resulting MS/MS data revealed several cellular proteins that are already known to interact with lactate dehydrogenase A (LDHA). Co-immunoprecipitation was performed to determine the interaction between LDHA and PPT1 in irradiated skin cells. Notably, ionizing radiation strengthened the binding of PPT1 with LDHA. Moreover, mechanistic insights into PPT1-mediated regulation of LDHA functions by PPT1 inhibitors. In addition, overexpression of GTP cyclohydrolase I (GCH1), the rate-limiting enzyme for BH4 synthesis decreased PPT1 expression, and restored NO production through BH4 treatment, suggesting that PPT1 expression was potentially regulated by NO. PPT1 was associated with the survival of irradiated skin cells. Taken together, this study reveals the molecular change of proteins with S-nitrosylation and the potential role of PPT1 in radiation-induced skin injury.