USP15 and Radiation-Induced DNA Damage Repair of Intestinal Epithelial Cells by Deubiquitinating and Stabilizing ATM

Purpose/Objective(s)

The intestine is vulnerable to structural and functional damage caused by exposure to radiation. Unfortunately, there is currently no effective prophylactic or therapeutic strategy available to mitigate radiation-induced intestinal injury (RIII). Deubiquitinating enzymes (DUBs) play a crucial role in repairing DNA breaks. Therefore, we conducted a new study on the pathogenesis of RIII by examining the role of DUBs, in order to identify potential directions for therapeutic or preventive measures in this area.

Materials/Methods

The effects of 14 Gy whole abdominal irradiation (WAI) on DUB levels in the intestine of C57BL/6J mice were investigated by RNA-seq analysis. In vivo and in vitro experiments were conducted using the ubiquitin-specific proteases 15 (USP15) inhibitor (USP15-IN-1). The impact of USP15 on the radiosensitization of HIEC-6 cells was observed. The survival and body weight of mice in each irradiated group were recorded, and the severity of radiation-induced intestinal injury (RIII) was evaluated through HE staining, immunohistochemistry (IHC), and the TUNEL method. USP15-bound proteins were identified and validated through mass spectrometry analysis. The role of USP15 in ataxia-telangiectasia mutated (ATM) deubiquitination and stability was determined by constructing a USP15 mutant (C298A). Finally, the potential reversal of USP15 knockdown’s promotional effects on radiosensitizing effect in HIEC-6 cells by ATM was investigated.

Results

USP15 is one of the top 20 highly expressed genes in the intestinal tissue of mice after exposure to 14Gy of WAI. Inhibition of USP15 resulted in increased radiosensitivity of HIEC-6 cells, as evidenced by a decrease in colony-forming ability and an increase in the formation of micronuclei and apoptotic cells, as well as an increase in 8-OHdG fluorescence intensity. Comet assay and γ-H2AX staining revealed more DNA damage in irradiated HIEC-6 cells treated with USP15-IN-1. In vivo, USP15 was found to modulate apoptosis and DNA damage in the small intestine of mice exposed to WBI. Mass spectrometry analysis showed that USP15 interacts with the protein kinase ATM, a key regulator of DNA double-strand break (DSB) signaling and stress response. By creating a mutant form of USP15(C298A), it was discovered that USP15 directly interacts with ATM, independent of its DUB activity, and can regulate the stability of ATM protein. Furthermore, USP15 was found to specifically disassemble K48-linked polyubiquitination of ATM but had no significant effect on monoubiquitination or other types of polyubiquitination. The radiosensitizing effect produced by knockdown of USP15 in HIEC-6 cells can be reversed by ATM.

Conclusion

Our findings demonstrate that USP15 plays a crucial role in repairing radiation damage in intestinal epithelial cells by counteracting ATM ubiquitination and degradation, which may be a novel mechanism involved in the occurrence and development of RIII.