Ubiquitin-specific protease 20(USP20) mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR

Abstract Background The severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) play pivotal roles in cardiac pathophysiology because of their precise regulation of protein function, localization and degradation.Objectives The objective of this study was to investigate the role and molecular mechanism of ubiquitin-specific peptidase 20 (USP20), a DUB, in doxorubicin-induced cardiotoxicity.Methods Cardiomyocyte-specific USP20-knockout (USP20-CKO) mice were utilized to assess the role of USP20 in doxorubicin-induced cardiomyopathy (DIC). Coimmunoprecipitation (co-IP) combined with liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) analysis was employed to screen the substrate protein of USP20. Furthermore, mutant plasmids of USP20 were constructed to elucidate the molecular mechanism underlying the regulation of human antigen R (HuR) by USP20. Finally, an AAV9 vector was used to overexpress USP20 in the hearts of cardiac-specific HuR-knockout mice to assess the interaction between USP20 and HuR.Results The results revealed a decrease in USP20 expression in Dox-stimulated mouse cardiomyocytes. Cardiomyocyte-specific USP20 knockout resulted in increased cardiomyocyte ferroptosis and led to DIC. Mechanistically, USP20 directly interacted with HuR through its ubiquitin-specific protease structural domain. Deubiquitination at position 154 was crucial for maintaining HuR protein stability by cleaving K48 ubiquitin chains and inhibiting proteasomal degradation. Additionally, HuR bound to GPX4 mRNA to suppress its degradation, thereby mitigating ferroptosis and contributing to alleviating DIC. Furthermore, targeted USP20 overexpression via AAV9 in cardiomyocytes significantly alleviated DIC. However, in mice with cardiomyocyte-specific HuR knockout, USP20 no longer had an anti-DIC effect, indicating that HuR, as a downstream target protein of USP20, plays an irreplaceable role in DIC.Conclusions Our findings indicate that USP20 enhances the stability of the HuR protein through deubiquitination, thereby inhibiting ferroptosis and mitigating DIC.